Isolation and Use of FAD2 and FAE1 From Camelina

ABSTRACT

The present invention provides isolated FAD2 and FAE1 genes and FAD2 and FAE1 protein sequences of  Camelina  species, e.g.,  Camelina sativa , mutations in  Camelina  FAD2 and FAE1 genes, and methods of using the same. In addition, methods of altering  Camelina  seed composition and/or improving  Camelina  seed oil quality are disclosed. Furthermore, methods of breeding  Camelina  cultivars and/or other closely related species to produce plants having altered or improved seed oil and/or meal quality are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 13/072,122, filed Mar. 25, 2011, now U.S. Pat. No. 9,035,131; whichclaims the benefit of U.S. Provisional Patent Application Ser. No.61/318,273, filed Mar. 26, 2010, and U.S. Provisional Patent ApplicationSer. No. 61/346,410, filed May 19, 2010. The contents of the aboveapplications are hereby incorporated by reference in their entiretiesfor all purposes.

TECHNICAL FIELD

The invention relates to the identification, isolation and use ofnucleic acid sequences, including genes, and nucleic acid fragmentsencoding fatty acid desaturase enzymes and/or fatty acid elongases,mutants thereof, and methods of altering lipid composition in Camelinaspecies, e.g., Camelina sativa.

BACKGROUND

The current concern about our global dependence on fossil fuels and theconsequent impact on climate change have brought biofuels to theforefront. This interest in biofuels has prompted researchers tocritically evaluate alternative feedstocks for biofuel production. Oneimportant, emerging biofuel crop is Camelina sativa L. Cranz(Brassicaceae), commonly referred to as “false flax” or“gold-of-pleasure”. Renewed interest in C. sativa as a biofuel feedstockis due in part to its drought tolerance and minimal requirements forsupplemental nitrogen and other agricultural inputs (Putnam, Budin etal. 1993; Zubr 1997; Gehringer, Friedt et al. 2006; Gugel and Falk2006). Similar to other non-traditional, renewable oilseed feedstockssuch as Jatropha curcas L. (“jatropha”), C. sativa grows on marginalland. Unlike jatropha, which is a tropical and subtropical shrub, C.sativa is native to Europe and is naturalized in North America, where itgrows well in the northern United States and southern Canada.

In addition to its drought tolerance and broad distribution, severalother aspects of C. sativa biology make it well suited for developmentas an oilseed crop. First, C. sativa is a member of the familyBrassicaceae, and thus is a relative of both the genetic model organismArabidopsis thaliana and the oilseed crop Brassica napus. The closerelationship between C. sativa and A. thaliana (Al-Shehbaz, Beilstein etal. 2006; Beilstein, Al-Shehbaz et al. 2006; Beilstein, Al-Shehbaz etal. 2008) makes the A. thaliana genome an ideal reference point for thedevelopment of genetic and genomic tools in C. sativa. Second, the oilcontent of C. sativa seeds is comparable to that of B. napus, rangingfrom 30 to 40% (w/w) (Budin, Breene et al. 1995; Gugel and Falk 2006),suggesting that agronomic lessons from the cultivation of B. napus areapplicable to C. sativa cultivation. Finally, the properties of C.sativa biodiesel are already well described (Rice, Frohlich et al. 1997;Frohlich and Rice 2005; Worgetter, Prankl et al. 2006), and both seedoil and biodiesel from C. sativa were used as fuel in engine trials withpromising results (Bernardo, Howard-Hildige et al. 2003; Frohlich andRice 2005).

The quality of a biodiesel, regardless of its source, is dependent uponthe fatty acid methyl ester (FAME) composition, which affects cold flowand oxidative stability (Knothe 2005; Durrett, Benning et al. 2008). Forexample, saturated FAMEs have poor cold flow properties since they canform crystals at lower temperatures, while the FAMEs frompolyunsaturated fatty acids remain in solution at colder temperatures,and thus have good cold flow properties (Stournas 1995; Serdari, Lois etal. 1999). In contrast, the relationship between saturation andoxidative stability is exactly opposite that of cold flow. Fatty acidsaturation is positively correlated with oxidative stability; saturatedfatty acids have the best oxidative stability and fatty acids with 2 orgreater double bonds have increasing oxidative instability (Knothe andDunn 2003; Knothe 2005; Durrett, Benning et al. 2008). Additionally,polyunsaturated FAMEs can result in increased NOx emissions, e.g., NO,NO₂ et al (McCormick, Graboski et al. 2001), and thus affect theproduction of a monitored pollutant. Very long chain fatty acids (VLCFA;as used herein, refers to those fatty acids containing greater than 18carbons) result in a biodiesel with a high distillation temperature thatdoes not meet existing standards (American Society for Testing andMaterials, ASTM), reducing marketability. Given these trade-offs, anideal biodiesel blend is high in oleic acid (18:1; carbons:doublebonds), low in polyunsaturated FAMEs, and with few long chain FAMEs.This blend is oxidatively stable, has a low cloud point, and meetsbiodiesel standards (ASTM; Knothe 2005; Durrett, Benning et al. 2008).

The naturally occurring oil composition of C. sativa negatively affectsits biodiesel properties. Polyunsaturated fatty acids such as linoleic(18:2) and alpha-linolenic (18:3) acids account for 52.1-54.7% of C.sativa seed oil (Ni Eidhin, Burke et al. 2003; Abramovic and Abram2005). This likely accounts for the low oxidative stability of C. sativaFAMEs (Bernardo, Howard-Hildige et al. 2003). C. sativa seeds alsocontain 21.4-22.4% VLCFA, of which 11-eicosenoic acid (20:1) at14.9-16.2% are especially abundant (Zubr 2002; Ni Eidhin, Burke et al.2003; Abramovic and Abram 2005), likely resulting in the highdistillation temperature of the FAMEs. Most desirable for biodiesel isoleic acid (18:1), which accounts for 14.0-17.4% of C. sativa seed oil(Budin, Breene et al. 1995; Zubr 2002; Ni Eidhin, Burke et al. 2003;Abramovic and Abram 2005). There is therefore the potential to optimizeCamelina oil for biodiesel production by decreasing both the amount ofpolyunsaturated fatty acids being produced from oleic acid anddecreasing the production of fatty acids with chain length of 18 carbonsor greater.

Genes affecting oil composition are well characterized in Arabidopsisthaliana, a close relative of Camelina sativa, as well as in some otherplants. For example, oleic acid (18:1) is converted to linoleic acid(18:2) in the endoplasmic reticulum by the membrane bounddelta-12-desaturase FATTY ACID DESATURASE 2 (FAD2). In Arabidopsis fad2mutants, levels of 18:1 oleic acid in the seeds increase by a factor of2-3.4 while levels of 18:2 linoleic acids are decreased by a factor of4-10 (Okuley, Lightner et al. 1994). Thus, mutations affecting FAD2 havebeen shown to lead to higher levels of oleic acid in A. thaliana andother studies have shown FAD2 has a similar role in crops such as canola(Hu, Sullivan-Gilbert et al. 2006), sunflower (Hongtrakul, Slabaugh etal. 1998) and peanut (Patel, Jung et al. 2004).

Very long chain fatty acids are formed in the cytosol of A. thaliana bysequential addition of 2 carbon units to 18 carbon fatty acid CoAconjugates. The rate limiting step is thought to be initialcondensation, catalyzed in the seed by FATTY ACID ELONGASE 1 (FAE1)(James Jr, Lim et al. 1995) (Kunst, Taylor et al. 1992). In wild typeArabidopsis, approximately 25% of fatty acids in seeds are long chainfatty acids, while fae1 mutants contain less than 1% long chain fattyacids. Interestingly, 18:1 content in seeds increases by a factor of 2in A. thaliana fae1 (Kunst, Taylor et al. 1992). In Brassica napus,reductions in long chain fatty acids, particularly erucic acid (22:1),are linked to changes in FAE1 activity (Han, Lühs et al. 2001; Katavic,Mietkiewska et al. 2002; Wang, Wang et al. 2008; Wu, et al. 2008).

The close relationship between A. thaliana and C. sativa suggests thatFAD2 and FAE1 may play similar roles in both species, making these genesgood targets for manipulation of oil composition in C. sativa. To ourknowledge, FAD2 and FAE1 gene sequences have not been previouslyreported for C. sativa. Indeed, published studies detailing the biologyof C. sativa and its closest relatives in the genus Camelina are few.However, several important findings can be drawn from the literature.Taxonomic treatments describe 11 species in the genus with a center ofdiversity in Eurasia (Akeroyd J: Camelina in Flora Europaea. 2nd edn.Cambridge, UK: Cambridge University Press; 1993.) although C. sativa, C.rumelica, C. microcarpa, and C. alyssum are naturalized weeds with broaddistributions. Camelina species can be annual or biennial, with somespecies requiring vernalization to induce flowering (Mirek Z: GenusCamelina in Poland—Taxonomy, Distribution and Habitats. FragmentaFloristica et Geobotanica 1981, 27:445-503.). Chromosome counts rangefrom n=6 in C. rumelica (Brooks R E: Chromosome number reports LXXXVIITaxon 1985, 34:346-351; Baksay L: The chromosome numbers andcytotaxonomical relations of some European plant species. Ann Hist-NatMus Natl Hung 1957:169-174.) or n=7 in C. hispida (Maassoumi A:Cruciferes de la flore d'Iran: etude caryosystematique. Thesis.Strasbourg, France, 1980.), upwards to n=20 in C. sativa, C. microcarpa,and C. alyssum (Gehringer A, Friedt W, Luhs W, Snowdon R J: Geneticmapping of agronomic traits in false flax (Camelina sativa subsp.sativa). Genome 2006, 49:1555-1563; Francis A, Warwick S: The Biology ofCanadian Weeds. 142. Camelina alyssum (Mill.) Thell.; C. microcarpaAndrz. ex D C.; C. sativa (L.) Crantz. Canadian Journal of Plant Science2009, 89:791-810.) Some Camelina species are interfertile; crosses of C.saliva with C. alyssum, and C. sativa with C. microcarpa, produce viableseed (Tedin O: Vererbung, Variation and Systematik in der GattungCamelina. Hereditas 1925, 6:19-386.) More recently, plastid simplesequence repeat (SSR) markers (Flannery M L, Mitchell F J, Coyne S,Kavanagh T A, Burke J I, Salamin N, Dowding P, Hodkinson T R: Plastidgenome characterisation in Brassica and Brassicaceae using a new set ofnine SSRs. Theor Appl Genet 2006, 113:1221-1231.) and randomly amplifiedpolymorphic DNA (RAPD) markers have been reported and a mapping studyusing amplified fragment length polymorphisms (AFLP) has been published(Gehringer A, Friedt W, Luhs W, Snowdon R J: Genetic mapping ofagronomic traits in false flax (Camelina sativa subsp. sativa). Genome2006, 49:1555-1563). Additionally, the sequences of a few C. sativatranscription factors are available from the literature (Martynov V V,Tsvetkov I L, Khavkin E E: Orthologs of arabidopsis CLAVATA 1 gene incultivated Brassicaceae plants. Ontogenez 2004, 35:41-46.) and inGenBank.

As an oilseed crop in the Brassicaceae family, Camelina sativa hasinspired renewed interest due to its potential for biofuelsapplications. Little is understood of the nature of the C. sativagenome, however. A study was undertaken by the present inventors tocharacterize two genes in the fatty acid biosynthesis pathway, fattyacid desaturase (FAD) 2 and fatty acid elongase (FAE) 1.

SUMMARY OF THE INVENTION

Camelina sativa is a re-emerging oilseed with tremendous potential as analternative biofuel crop and for which genomic information is becomingincreasingly available. The inventors have characterized two genesencoding fatty acid biosynthesis enzymes and, in the process, havediscovered unexpected complexity in the C. sativa genome.

The present inventors disclose herewith the sequences of three copies ofboth FAE1 and FAD2 recovered from C. sativa. Southern blots were used todetermine whether the recovered copies are allelic or if they representmultiple loci. Moreover, the inventors performed phylogenetic analysesto infer the evolutionary history of the copies, and quantitative PCR(qPCR) to explore whether there is evidence of functional divergenceamong them. To better understand the C. sativa genome and to determinewhether the multiple copies recovered are the result ofpolyploidization, the inventors also analyzed the genome sizes of C.sativa and its closest relatives in the genus Camelina by flowcytometry. Collectively the inventors' results indicate that C. sativais an allohexaploid whose oil composition is likely influenced by morethan one functional copy of FAE1 and FAD2. This should allow highlyspecialized blends of oil to be produced from C. sativa with mutationsin FAE1 and FAD2, greatly increasing the utility of this emergingbiofuel crop.

The present inventors unexpectedly discovered by Southern analysis thatin C. sativa, there are three copies of both FAD2 and FAE1 as well asLFY, a known single copy gene in other species. All three copies of bothFAD2 and FAE1 are expressed in developing seeds, and sequence alignmentsshow that previously described conserved sites are present, suggestingthat all three copies of both genes could be functional. The regionsdownstream of FAD2 and upstream of FAE1 demonstrate co-linearity withthe Arabidopsis genome. In addition, results from flow cytometryindicate that the DNA content of C. sativa is approximately three-foldthat of diploid Camelina relatives. Phylogenetic analyses furthersupport a history of duplication and indicate that C. saliva and C.microcarpa might share a parental genome. FAD2 and FAE1 sequences fromspecies in the tribe of Camelineae have been deposited in Genbank at theNCBI [Genbank: GU929417-GU929441, SEQ ID NOs: 1 to 6, and SEQ ID NOs45-63, as listed below, which are incorporated by reference in theirentireties.

GenBank SEQ access # Sequence Name ID NO GU929417 Camelina sativa FAD2 A(upstream, coding and downstream genomic 1 sequence) GU929418 Camelinasativa FAD2 B (upstream, coding and downstream genomic 2 sequence)GU929419 Camelina sativa FAD2 C (upstream, coding and downstream genomic3 sequence) GU929420 Camelina sativa FAE1 A [upstream gene (KCS17),intergenic region and 4 coding) GU929421 Camelina sativa FAEI B(upstream gene (KCS17), intergenic region and 5 coding) GU929422Camelina sativa FAE1 C [upstream gene (KCS17), intergenic region and 6coding) GU929423 Capsella rubella FAD2 45 GU929424 Arabidopsis lyrataFAD2 46 GU929425 Arabidopsis lyrata FAE1 47 GU929426 Camelina hispidaFAD2 48 GU929427 Camelina hispida FAE1-1 49 GU929428 Camelina hispidaFAE1-2 50 GU929429 Camelina laxa FAD2 51 GU929430 Camelina laxa FAE1-152 GU929431 Camelina laxa FAE1-2 53 GU929432 Camelina microcarpa FAD2 A54 GU929433 Camelina microcarpa FAD2 B 55 GU929434 Camelina microcarpaFAD2 C 56 GU929435 Camelina microcarpa FAE1 A 57 GU929436 Camelinamicrocarpa FAE1 B 58 GU929437 Camelina microcarpa FAE1 C 59 GU929438Camelina rumelica FAD2-1 60 GU929439 Camelina rumelica FAD2-2 61GU929440 Camelina rumelica FAE1-1 62 GU929441 Camelina rumelica FAE1-263

The C. sativa genome appears to be organized in three copies, and can beconsidered to be an allohexaploid. The discovery of triplication anddivergence of genes that in known diploids are present in single copy,the cytometrically determined genome size of Camelina species, thepattern of relationship and inferred duplication history in the genetrees, together with the previously known chromosome counts for thistaxon, indicate a likely allohexaploid genomic constitution. Thecharacterization of genes encoding key functions of fatty acidbiosynthesis lays the foundation for future manipulations of thispathway in Camelina sativa, which allows for the future manipulation ofoil composition of this emerging biofuel crop.

The present invention provides an isolated nucleic acid sequencecomprising a sequence selected from the group consisting of SEQ ID NOs:1 to 6 and 45 to 63, and fragments and variations derived from thereof,which encode a plant fatty acid synthesis gene.

In one embodiment, the present invention provides an isolatedpolynucleotide encoding plant fatty acid desaturase, comprising anucleic acid sequence that shares at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, atleast 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least99.7%, at least 99.8%, or at least 99.9% identity to SEQ ID NO: 1, 2, 3,45, 46, 48, 51, 54, 55, 56, 60, and/or 61.

In another embodiment, the present invention provides an isolatedpolynucleotide encoding fatty acid elongase, comprising a nucleic acidsequence that shares at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, atleast 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least99.8%, or at least 99.9% identity to SEQ ID NO: 4, 5, 6, 47, 49, 50, 52,53, 57, 58, 59, 62, and/or 63.

The present invention further provides an isolated amino acid sequence(e.g., a peptide, polypeptide and the like) comprising a sequenceselected from the group consisting of SEQ ID NOs: 7 to 12, and fragmentsand variations derived from thereof, which form a plant fatty acidsynthesis protein.

In some embodiments, the present invention provides an isolated aminoacid sequence which forms a protein that shares an amino acid sequencehaving at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, atleast 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least99.9% identity to SEQ ID NO: 7, 8, 9, 64, 65, 67, 70, 73, 74, 75, 79,and/or 80.

In one embodiment, the present invention provides an isolated amino acidsequence which forms a protein that shares an amino acid having at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, at least99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%,at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%identity to SEQ ID NO: 10, 11, 12, 66, 68, 69, 71, 72, 76, 77, 78, 81,and/or 82.

The present invention also provides a chimeric gene comprising theisolated nucleic acid sequence of any one of the polynucleotidesdescribed above operably linked to suitable regulatory sequences.

The present invention also provides a recombinant construct comprisingthe chimeric gene as described above.

The present invention further comprises interfering RNA (RNAi) based onthe expression of the nucleic acid sequences of the present invention,wherein such RNAi includes but is not limited to microRNA (miRNA) andsmall interfering RNA (siRNA) which can be used in gene silencingconstructs.

The present invention also provides a transformed host cell comprisingthe chimeric gene as described above. In one embodiment, said host cellis selected from the group consisting of bacteria, yeasts, filamentousfungi, algae, animals, and plants.

The present invention in another aspect, provides a plant comprising inits genome one or more genes as described herein, one or more genes withmutations as described herein, or the chimeric genes as describedherein.

The present invention in another aspect, provides a plant seed obtainedfrom the plants described herein, wherein the plants comprise in theirgenomes one or more genes as described herein, one or more genes withmutations as described herein, or the chimeric genes as describedherein.

The present invention in another aspect, provides Camelina oil obtainedfrom the seeds of a Camelina plant comprising the one or more genesdescribed herein, one or more genes with mutations as described herein,or one or more chimeric genes as described herein.

The present invention in another aspect, provides meals made fromCamelina plants comprising the one or more genes described herein, oneor more genes with mutations as described herein, or one or morechimeric genes as described herein. In some embodiments, the meal is abyproduct of the extraction of the oil from said Camelina seeds. In someembodiments, said Camelina plant has reduced level of erucic acid (22:1)compared to a wild type Camelina plant. In some embodiments, saidCamelina plant has less than 4%, less than 3%, less than 2%, less than1%, or less than 0.1% erucic acid (22:1) compared to the wild type. Infurther embodiments, the Camelina meal is included in the diets of ananimal for about 0.1%, about 0.5%, about 1%, about 2%, about 3%, about4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% oftheir feed on a weight or volume basis.

Thus, the present invention provides methods of altering and/orimproving Camelina fatty acids composition by disrupting and/or alteringone, two, or all three copies of one or more fatty acid synthesis genesin Camelina. Methods of disrupting and/or altering gene function includebut are not limited to mutagenesis (e.g., chemical mutagenesis,radiation mutagenesis, transposon mutagenesis, insertional mutagenesis,signature tagged mutagenesis, site-directed mutagenesis, and naturalmutagenesis), antisense, knock-outs, and/or RNA interference.

In some embodiments, the methods comprise introducing mutations in oneor more FAD2 genes and/or one or more FAE1 genes of Camelina. In someembodiments, the methods disclosed herein comprise utilizing Camelinamutants with mutations in all three FAD2 genes (e.g., FAD2 A, FAD2 B,and FAD2 C), and/or Camelina mutants with mutations in all three FAE1genes (e.g., FAE1 A, FAE1 B, and FAE1 C).

The present invention provides mutants in FAD2 A, FAD2 B, FAD2 C, FAE1A, FAE1 B, and FAE1 C, including but not limited to those as listed inTables 7-12. In some embodiments, the methods of altering and/orimproving Camelina fatty acids composition comprise utilizing one ormore Camelina mutants for any one or more of the mutations listed inTables 7 to 12 and as described in Example 11. In some embodiments,mutations in one or more copies of FAD2 genes and/or mutations in one ormore copies of FAE1 genes as described in the Tables 7 to 12 areintegrated together to create mutant plants with double, triple,quadruple et al. mutations in one, two, or all three copies of FAD2and/or FAE1 genes. In some embodiments, the mutations described in theTables 7-12 can be integrated into Camelina sativa cultivars other thanCs32 (commercial name as 5030) or other Camelina species by classicbreeding methods, with or without the help of marker-facilitatedinter-cultivar gene transfer methods. In some embodiments, mutationsdescribed in the Tables 7-12 can be integrated into species closelyrelated to Camelina sativa. In still other embodiments, amino acids inconserved domains or sites compared to FAD2 or FAE1 orthologs in otherspecies can be substituted or deleted to make mutants with reduced orabolished activity, mutants that lead to loss-of-function (e.g., proteininstability), and/or mutants that lead to gain-of-function (e.g., morestable or more active protein).

In some embodiments, one, two, or all three copies of Camelina FAD2and/or FAE1 genes, and one, two, or all three copies of other non-FAD,non-FAE fatty acid synthesis genes are disrupted. In still someembodiments, one, two, or all three copies of Camelina FAD2 and/or FAE1genes are disrupted, while one or more non-FAD, non-FAE fatty acidsynthesis genes are overexpressed. In still more embodiments, one, two,or all three copies of Camelina FAD2 and/or FAE1 genes are disrupted,while one or more non-fatty-acid-synthesis genes are disrupted and/oroverexpressed.

In another aspect, the present invention provides methods of producingCamelina seed oil containing altered and/or increased levels of oleicacid (18:1), and/or altered or reduced levels of polyunsaturated fattyacids, and/or decreased very long chain fatty acids. Such methodscomprising utilizing the Camelina plants comprising the chimeric genesas described herein, or Camelina plants with disrupted FAD2 and/or FAE1genes as described herein. As used herein, the phrase “very long chainfatty acid” refers to a fatty acid with more than 18 carbons.

The present invention also provides methods of increasing the activityof a FAD2 and/or FAE1 protein in a Camelina plant cell, plant part,tissue culture or whole plant comprising transforming the plant cell,plant part, tissue culture or whole plant with a chimeric genecomprising one FAD2 and/or FAE1 gene encoding the polypeptide of thepresent invention, or functional variants thereof. In one embodiment,the chimeric gene is overexpressed. As used herein, a functional variantof a protein refers to a polypeptide comprising one or more amino acidmodifications (e.g., substitution, deletion, modification, et al)compared to the original protein, but still maintains the activity ofthe original protein. In the present invention, “overexpressionpromoter” means a promoter capable of causing strong expression (largeamount expression) of a gene that has been ligated thereto in host plantcells. The overexpression promoter of the present invention may beeither an inducible promoter or a constitutive promoter. A promoter is aDNA comprising an expression control region generally located on the 5′upstream of a structural gene or a modified sequence thereof. In thepresent invention, any promoters appropriate for foreign gene expressionin plant cells can be used as overexpression promoters. Non-limitingexamples of such overexpression promoters to be used in the presentinvention include, but are not limited to, a cauliflower mosaic virus(CaMV) 35S promoter, a rice actin promoter, a modified 35S promoter, oran embryo-specific promoter. As used herein an “embryo-specificpromoter” refers to a promoter of an embryo-specific gene. Anembryo-specific gene is preferentially expressed during embryodevelopment in a plant. For purposes of the present disclosure, embryodevelopment begins with the first cell divisions in the zygote andcontinues through the late phase of embryo development (characterized bymaturation, desiccation, dormancy), and ends with the production of amature and desiccated seed. Embryo-specific genes can be furtherclassified as “early phase-specific” and “late phase-specific”. Earlyphase-specific genes are those expressed in embryos up to the end ofembryo morphogenesis. Late phase-specific genes are those expressed frommaturation through to production of a mature and desiccated seed. Anearly phase-specific promoter is a promoter that initiates expression ofa protein prior to day 7 after pollination in Arabidopsis or anequivalent stage in another plant species. Non-limiting examples ofpromoter sequences that can be used in the present invention include apromoter for the amino acid permease gene (AAP1) (e.g., the AAP1promoter from Arabidopsis thaliana, Hirner et al, Plant J. 14:535-544,1998), a promoter for the oleate 12-hydroxylase:desaturase gene (e.g.,the promoter designated LFAH 12 from Lesquerellafendleri, Broun et al,Plant J. 13:201-210, 1998), a promoter for the 2S2 albumin gene (e.g.,the 2S2 promoter from Arabidopsis thaliana, Guerche et al, Plant cell2:469-478, 1990), a fatty acid elongase gene promoter (FAE1) (e.g., theFAE1 promoter from Arabidopsis thaliana, Rossak et al, Plant MoI Biol.46:717-715, 2001), and the leafy cotyledon gene promoter (LEC2) (e.g.,the LEC2 promoter from Arabidopsis thaliana, Kroj et al Development130:6065-6073, 2003). Other early embryo-specific promoters of interestinclude, but are not limited to, seedstick (Pinyopich et al, Nature424:85-88, 2003), Fbp7 and Fbpl 1 (Petunia Seedstick) (Colombo et al,Plant Cell. 9:703-715, 1997), Banyuls (Devic et al, Plant J. 19:387-398,1999), agl-15 and agl-18 (Lehti-Shiu et al, Plant MoI Biol. 58:89-107,2005), Phel (Kohler et al, Genes Develop. 17:1540-1553, 2003), Perl(Haslekas et al, Plant MoI Biol. 36:833-845, 1998; Haslekas et al, PlantMoI Biol. 53:313-326, 2003), embl75 (Cushing et al, Planta 221:424-436,2005), LIl (Kwong et al, Plant Cell 15:5-18, 2003), Lecl (Lotan et al,Cell 93:1195-1205, 1998), Fusca3 (Kroj et al, Development 130:6065-6073,2003), ttl2 (Debeaujon et al, Plant Cell 13:853-871, 2001), ttl6 (Nesiet al, Plant Cell 14:2463-2479, 2002), A-RZf (Zou and Taylor, Gene196:291-295, 1997), TtGl (Walker et al, Plant Cell 11:1337-1350, 1999;Tsuchiya et al, Plant J. 37:73-81, 2004), TtI (Sagasser et al, GenesDev. 16:138-149, 2002), TT8 (Nesi et al, Plant Cell 12:1863-1878, 2000),Gea-8 (carrot) (Lin and Zimmerman, J. Exp. Botany 50:1139-1147, 1999),Knox (rice) (Postma-Haarsma et al, Plant MoI. Biol. 39:257-271, 1999),Oleosin (Plant et al, Plant MoI Biol. 25:193-205, 1994; Keddie et al,Plant MoI Biol. 24:121-14$, 1994), ABI3 (Ng et al, Plant MoI Biol.54:25-38, 2004; Parcy et al, Plant Cell 6:1567-1582, 1994), and thelike.

The present invention also provides methods of decreasing the activityof a FAD2 and/or FAE1 protein in a Camelina plant cell, plant part,tissue culture or whole plant comprising contacting the plant cell,plant part, tissue culture or whole plant with an inhibitory nucleicacid having complementarity to a gene encoding the FAD2 and/or FAE1protein.

In one aspect, the present invention provides methods of breedingCamelina species producing altered levels of fatty acids in the seed oiland/or meal. In one embodiment, such methods comprise making a crossbetween a Camelina mutant with one or more mutations listed in Tables7-12 with a second Camelina cultivar to produce an F1 plant;backcrossing the F1 plant to the second Camelina cultivar; and repeatingthe backcrossing step to generate an near isogenic line, wherein the oneor more mutations are integrated into the genome of the second Camelinacultivar; wherein the near isogenic line derived from the secondCamelina cultivar with the integrated mutations has altered seed oilcomposition. Optionally, such methods can be facilitated by molecularmarkers.

In another aspect, the present invention provides methods of breedingspecies close to Camelina sativa, wherein said species produces alteredlevels of fatty acids in the seed oil and/or meal. For example,intertribal somatic hybridizations are possible between C. sativa and B.oleracea (see, e.g., Lise N. Hansen, 1998, Euphytica, Volume 104, No. 3,pages 173-179). In one embodiment, such methods comprise making a crossbetween the Camelina mutants with one or more mutations listed in Tables7-12 with a species that is closely related to the Camelina speciescontaining the mutations to make an F1 plant; backcrossing the F1 plantsto the species that is closely related to the Camelina speciescontaining the mutations; and, repeating backcrossing step to generatean near isogenic line, wherein the one or more mutations are integratedinto the genome of the species that is closely related to the Camelinaspecies containing the mutations; wherein the near isogenic line derivedfrom the species that is closely related to the Camelina speciescontaining the mutations has integrated these mutations and has alteredseed oil composition. Optionally, such method can be facilitated bymolecular markers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C depict Southern blot analysis of Camelina sativa andArabidopsis thaliana. A blot containing genomic DNA from C. sativa andA. thaliana digested with EcoRI or a combination of EcoRI and BamHI washybridized with an α-32 P dCTP—labeled (1A) FAD2 probe, (1B) FAE1 probeor (1C) LFY probe obtained from PCR amplification of C. sativa DNA.

FIGS. 2A-2B depict FAD2 and FAE1 protein alignment. FIG. 2A shows aminoacid sequence comparison of the three Camelina sativa FAD2 sequences,Arabidopsis thaliana FAD2 sequence [Genbank: NM_(—)112047], Brassicarapa FAD2 sequence [Genbank: AJ459107], Glycine max FAD2-3 sequence[Genbank: DQ532371], Zea mays FAD2 sequence [Genbank: AB257309]. Blueunderlines below the sequences indicate amino acids conserved in all 50FAD2 sequences (Belo, Zheng et al. 2008) while the green underlineindicates the ER localization signal (McCartney, Dyer et al. 2004). Thethree His boxes described by Tocher D R (1998) are indicated with redboxes. FIG. 2B shows amino acid sequence comparison of the threeCamelina sativa FAE1 sequences, Arabidopsis thaliana FAE1 [Genbank:NM_(—)119617], Crambe abyssinica [Genbank: AAX22298], Brassica rapa HEACFAE1 [Genbank: Y14975], Brassica rapa LEAC FAE1 [Genbank: Y14974],Limnanthes alba (meadow foam) [Genbank: AF247134], Tropaeolum majus(nasturtium) [Genbank: ABD77097]. Blue underlines below the sequenceindicate the asparagine at position 424 and the highly conservedhistidine and cysteine residues described by Ghanevati and Jaworski(Ghanevati and Jaworski 2001; Ghanevati and Jaworski 2002). The red boxindicates the region highly conserved among condensing enzymes in verylong chain fatty acid biosynthesis (Moon, Smith et al. 2001)Abbreviations: Heac=High erucic acid, Leac=Low erucic acid.

FIGS. 3A-3D depict FAD2 and FAE1 Expression in Developing Seeds.Relative combined expression of all three copies of (3A) FAD2 and (3B)FAE1 measured by real time quantitative PCR at 15, 20, 25, and 30 dayspost anthesis (DPA) and in 2 week old seedlings. The 20 DPA sample,which expressed FAD2 and FAE1 at the highest amount, was used as thecalibrator. Error bars represent the standard deviation of 3 replicateexperiments. Sequenom SNP analysis demonstrating the expression of eachversion of (3C) FAD2 or (3D) FAE1 relative to the other versions. Errorbars represent the standard deviation of three (for FAD2) or four (forFAE1) SNP analyses. Because FAE1 is not expressed in C. sativa seedlings(3B), the relative expression of the 3 copies of FAE1 in seedling tissueis not shown (3D).

FIG. 4 depicts structure and conservation of the KCS17-FAE1 intergenicregion in Camelina sativa. The three putative homologous regions inallohexaploid C. sativa are aligned to the orthologous region ofArabidopsis to display blocks of homology identified on a dot matrix byperfect conservation of a sliding window of 9 bases. The KCS17 and FAE1gene, respectively blue and red, flank a variable region in whichconserved blocks common to two or more genomes are marked by differentshades of brown. Lined regions display reduced or no conservation. Thelarge variation in the intergenic region of the triplicated KCS17-FAE1DNA of C. sativa is consistent with independent evolution before reunionof diverged genomes by allohexaploidization.

FIG. 5 depicts genome content of Camelina species. 1C nuclei werestained with propidium iodide and analyzed by flow cytometry. Error barsrepresent the standard deviation of 2-4 replicate samples.

FIGS. 6A-6B depict phylogenetic analyses of Camelineae FAD2 and FAE1.Maximum-likelihood trees showing branch length and bootstrap support(100 bootstrap replicates) for (6A) 15 FAD2 sequences from species fromthe tribe Camelineae calculated using the TVM+I+Γ model in PAUP* androoted with Brassica rapa FAD2 (−LnL 3665.277); and for (6B) 15 FAE1sequences from species from the tribe Camelineae calculated using theHKY+I+Γ model in PAUP* and rooted with Crambe abyssinica FAE1 (−LnL5051.552). Sequences obtained from Genbank are Capsella bursa-pastorisFAD2 [Genbank: DQ518293], Arabidopsis thaliana FAD2 [Genbank:NM_(—)112047], Brassica rapa FAD2 [Genbank: AJ459107], Arabidopsisthaliana FAE1 [Genbank: NM_(—)119617], and Crambe abyssinica FAE1[Genbank: AY793549].

FIG. 7 depicts a simplified version of fatty acid synthesis pathways inplant.

FIGS. 8A-8B depict an exemplary field growth of EMS mutagenized CamelinaM2 population (upper-panel (8A)), and exemplary mutant M2 plants withmorphological changes (lower-panel (8B)).

FIG. 9 depicts an exemplary LI-COR® gel identifying mutants in CamelinaFAD2 genes.

FIG. 10 depicts proximate locations of mutations in FAD2 A and B, whichwere used in the preliminary GC analysis. “H” identifies a His box.

FIG. 11 depicts a representative composition of Camelina sativa seedoil.

FIGS. 12A-12B depict fatty acid compositions in FAD2 mutants (12A) andin FAE1 mutants (12B) measured by gas chromatography. Note: FIG. 12B issummarized FAE1 data from GC test 4 and replaces FIG. 14B from U.S.Provisional Application No. 61/318,273, which summarized FAE1 data fromGC test 3.

FIG. 13 depicts lipid synthesis in the plastid and cytoplasm ofoilseeds. Key enzymes are in red text and boxed. ACCase=acetyl co-Acarboxylase, KAS=β-ketoacyl-acyl carrier protein (ACP) synthase,GPAT=glycerol phosphate acyltransferase, LPAAT=lysophosphatidic acidacyltransferase, PAP=phosphatidate phosphatase, DAGAT=diacylglycerolacyltransferase, R=fatty acyl group, P=phosphate group, CPT=chloroplast

DETAILED DESCRIPTION Definition

As used herein, the verb “comprise” as is used in this description andin the claims and its conjugations are used in its non-limiting sense tomean that items following the word are included, but items notspecifically mentioned are not excluded.

As used herein, the term “plant” refers to any living organism belongingto the kingdom Plantae (i.e., any genus/species in the Plant Kingdom).This includes familiar organisms such as but not limited to trees,herbs, bushes, grasses, vines, ferns, mosses and green algae. The termrefers to both monocotyledonous plants, also called monocots, anddicotyledonous plants, also called dicots. Examples of particular plantsinclude but are not limited to corn, potatoes, roses, apple trees,sunflowers, wheat, rice, bananas, tomatoes, opo, pumpkins, squash,lettuce, cabbage, oak trees, guzmania, geraniums, hibiscus, clematis,poinsettias, sugarcane, taro, duck weed, pine trees, Kentucky bluegrass, zoysia, coconut trees, brassica leafy vegetables (e.g. broccoli,broccoli raab, Brussels sprouts, cabbage, Chinese cabbage (Bok Choy andNapa), cauliflower, cavalo, collards, kale, kohlrabi, mustard greens,rape greens, and other brassica leafy vegetable crops), bulb vegetables(e.g. garlic, leek, onion (dry bulb, green, and Welch), shallot, andother bulb vegetable crops), citrus fruits (e.g. grapefruit, lemon,lime, orange, tangerine, citrus hybrids, pummelo, and other citrus fruitcrops), cucurbit vegetables (e.g. cucumber, citron melon, edible gourds,gherkin, muskmelons (including hybrids and/or cultivars of cucumismelons), water-melon, cantaloupe, and other cucurbit vegetable crops),fruiting vegetables (including eggplant, ground cherry, pepino, pepper,tomato, tomatillo, and other fruiting vegetable crops), grape, leafyvegetables (e.g. romaine), root/tuber and corm vegetables (e.g. potato),and tree nuts (almond, pecan, pistachio, and walnut), berries (e.g.,tomatoes, barberries, currants, elderberries, gooseberries,honeysuckles, mayapples, nannyberries, Oregon-grapes, see-buckthorns,hackberries, bearberries, lingonberries, strawberries, sea grapes,lackberries, cloudberries, loganberries, raspberries, salmonberries,thimbleberries, and wineberries), cereal crops (e.g., corn, rice, wheat,barley, sorghum, millets, oats, ryes, triticales, buckwheats, fonio, andquinoa), pome fruit (e.g., apples, pears), stone fruits (e.g., coffees,jujubes, mangos, olives, coconuts, oil palms, pistachios, almonds,apricots, cherries, damsons, nectarines, peaches and plums), vine (e.g.,table grapes, wine grapes), fibber crops (e.g. hemp, cotton),ornamentals, and the like. For example, the plant is a species in thetribe of Camelineae, such as C. alyssum, C. anomala, C. grandiflora, C.hispida, C. laxa, C. microcarpa, C. microphylla, C. persistens, C.rumelica, C. sativa, C. Stiefelhagenii, or any hybrid thereof.

As used herein, the term “plant part” refers to any part of a plantincluding but not limited to the shoot, root, stem, seeds, stipules,leaves, petals, flowers, ovules, bracts, branches, petioles, internodes,bark, pubescence, tillers, rhizomes, fronds, blades, pollen, stamen, andthe like. The two main parts of plants grown in some sort of media, suchas soil, are often referred to as the “above-ground” part, also oftenreferred to as the “shoots”, and the “below-ground” part, also oftenreferred to as the “roots”.

The term “a” or “an” refers to one or more of that entity; for example,“a gene” refers to one or more genes or at least one gene. As such, theterms “a” (or “an”), “one or more” and “at least one” are usedinterchangeably herein. In addition, reference to “an element” by theindefinite article “a” or “an” does not exclude the possibility thatmore than one of the elements are present, unless the context clearlyrequires that there is one and only one of the elements.

As used herein, the term “chimeric protein” refers to a construct thatlinks at least two heterologous proteins into a single macromolecule(fusion protein).

As used herein, the term “nucleic acid” refers to a polymeric form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides, or analogs thereof. This term refers to theprimary structure of the molecule, and thus includes double- andsingle-stranded DNA, as well as double- and single-stranded RNA. It alsoincludes modified nucleic acids such as methylated and/or capped nucleicacids, nucleic acids containing modified bases, backbone modifications,and the like. The terms “nucleic acid” and “nucleotide sequence” areused interchangeably.

As used herein, the terms “polypeptide,” “peptide,” and “protein” areused interchangeably herein to refer to polymers of amino acids of anylength. These terms also include proteins that are post-translationallymodified through reactions that include glycosylation, acetylation andphosphorylation.

As used herein, the term “homologous” or “homolog” or “ortholog” isknown in the art and refers to related sequences that share a commonancestor or family member and are determined based on the degree ofsequence identity. The terms “homology”, “homologous”, “substantiallysimilar” and “corresponding substantially” are used interchangeablyherein. They refer to nucleic acid fragments wherein changes in one ormore nucleotide bases do not affect the ability of the nucleic acidfragment to mediate gene expression or produce a certain phenotype.These terms also refer to modifications of the nucleic acid fragments ofthe instant invention such as deletion or insertion of one or morenucleotides that do not substantially alter the functional properties ofthe resulting nucleic acid fragment relative to the initial, unmodifiedfragment. It is therefore understood, as those skilled in the art willappreciate, that the invention encompasses more than the specificexemplary sequences. These terms describe the relationship between agene found in one species, subspecies, variety, cultivar or strain andthe corresponding or equivalent gene in another species, subspecies,variety, cultivar or strain. For purposes of this invention homologoussequences are compared. “Homologous sequences” or “homologs” or“orthologs” are thought, believed, or known to be functionally related.A functional relationship may be indicated in any one of a number ofways, including, but not limited to: (a) degree of sequence identityand/or (b) the same or similar biological function. Preferably, both (a)and (b) are indicated. The degree of sequence identity may vary, but inone embodiment, is at least 50% (when using standard sequence alignmentprograms known in the art), at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least about 91%,at least about 92%, at least about 93%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least 98.5%, or at least about 99%, or at least 99.5%, or at least99.8%, or at least 99.9%. Homology can be determined using softwareprograms readily available in the art, such as those discussed inCurrent Protocols in Molecular Biology (F. M. Ausubel et al., eds.,1987) Supplement 30, section 7.718, Table 7.71. Some alignment programsare MacVector (Oxford Molecular Ltd, Oxford, U.K.) and ALIGN Plus(Scientific and Educational Software, Pennsylvania). Other non-limitingalignment programs include Sequencher (Gene Codes, Ann Arbor, Mich.),AlignX, and Vector NTI (Invitrogen, Carlsbad, Calif.).

As used herein, the term “nucleotide change” or “nucleotidemodification” refers to, e.g., nucleotide substitution, deletion, and/orinsertion, as is well understood in the art. For example, mutationscontaining alterations that produce silent substitutions, additions, ordeletions, but do not alter the properties or activities of the encodedprotein or how the proteins are made.

As used herein, the term “protein modification” refers to, e.g., aminoacid substitution, amino acid modification, deletion, and/or insertion,as is well understood in the art.

As used herein, the term “derived from” refers to the origin or source,and may include naturally occurring, recombinant, unpurified, orpurified molecules. A nucleic acid or an amino acid derived from anorigin or source may have all kinds of nucleotide changes or proteinmodification as defined elsewhere herein.

As used herein, the term “at least a portion” of a nucleic acid orpolypeptide means a portion having the minimal size characteristics ofsuch sequences, or any larger fragment of the full length molecule, upto and including the full length molecule. For example, a portion of anucleic acid may be 12 nucleotides, 13 nucleotides, 14 nucleotides, 15nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19nucleotides, 20 nucleotides, 22 nucleotides, 24 nucleotides, 26nucleotides, 28 nucleotides, 30 nucleotides, 32 nucleotides, 34nucleotides, 36 nucleotides, 38 nucleotides, 40 nucleotides, 45nucleotides, 50 nucleotides, 55 nucleotides, and so on, going up to thefull length nucleic acid. Similarly, a portion of a polypeptide may be 4amino acids, 5 amino acids, 6 amino acids, 7 amino acids, and so on,going up to the full length polypeptide. The length of the portion to beused will depend on the particular application. A portion of a nucleicacid useful as hybridization probe may be as short as 12 nucleotides; inone embodiment, it is 20 nucleotides. A portion of a polypeptide usefulas an epitope may be as short as 4 amino acids. A portion of apolypeptide that performs the function of the full-length polypeptidewould generally be longer than 4 amino acids.

As used herein, “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences includes reference to the residuesin the two sequences which are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. Where sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences which differ by suchconservative substitutions are said to have “sequence similarity” or“similarity.” Means for making this adjustment are well-known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., according to the algorithm of Meyersand Miller, Computer Applic. Biol. Sci., 4:11-17 (1988).

As used herein, the term “suppression” or “disruption” of regulationrefers to reduced activity of regulatory proteins, and such reducedactivity can be achieved by a variety of mechanisms including antisense,mutation knockout or RNAi. Antisense RNA will reduce the level ofexpressed protein resulting in reduced protein activity as compared towild type activity levels. A mutation in the gene encoding a protein mayreduce the level of expressed protein and/or interfere with the functionof expressed protein to cause reduced protein activity.

As used herein, the terms “polynucleotide”, “polynucleotide sequence”,“nucleic acid sequence”, “nucleic acid fragment”, and “isolated nucleicacid fragment” are used interchangeably herein. These terms encompassnucleotide sequences and the like. A polynucleotide may be a polymer ofRNA or DNA that is single- or double-stranded, that optionally containssynthetic, non-natural or altered nucleotide bases. A polynucleotide inthe form of a polymer of DNA may be comprised of one or more segments ofcDNA, genomic DNA, synthetic DNA, or mixtures thereof. Nucleotides(usually found in their 5′-monophosphate form) are referred to by asingle letter designation as follows: “A” for adenylate ordeoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate ordeoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uridylate,“T” for deoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines(C or T), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N”for any nucleotide.

The term “primer” as used herein refers to an oligonucleotide which iscapable of annealing to the amplification target allowing a DNApolymerase to attach, thereby serving as a point of initiation of DNAsynthesis when placed under conditions in which synthesis of primerextension product is induced, i.e., in the presence of nucleotides andan agent for polymerization such as DNA polymerase and at a suitabletemperature and pH. The (amplification) primer is preferably singlestranded for maximum efficiency in amplification. Preferably, the primeris an oligodeoxyribonucleotide. The primer must be sufficiently long toprime the synthesis of extension products in the presence of the agentfor polymerization. The exact lengths of the primers will depend on manyfactors, including temperature and composition (A/T and G/C content) ofprimer. A pair of bi-directional primers consists of one forward and onereverse primer as commonly used in the art of DNA amplification such asin PCR amplification.

As used herein, “coding sequence” refers to a DNA sequence that codesfor a specific amino acid sequence. “Regulatory sequences” refer tonucleotide sequences located upstream (5′ non-coding sequences), within,or downstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence.

As used herein, “regulatory sequences” may include, but are not limitedto, promoters, translation leader sequences, introns, andpolyadenylation recognition sequences.

As used herein, “promoter” refers to a DNA sequence capable ofcontrolling the expression of a coding sequence or functional RNA. Thepromoter sequence consists of proximal and more distal upstreamelements, the latter elements often referred to as enhancers.Accordingly, an “enhancer” is a DNA sequence that can stimulate promoteractivity, and may be an innate element of the promoter or a heterologouselement inserted to enhance the level or tissue-specificity of apromoter. Promoters may be derived in their entirety from a native gene,or be composed of different elements derived from different promotersfound in nature, or even comprise synthetic DNA segments. It isunderstood by those skilled in the art that different promoters maydirect the expression of a gene in different tissues or cell types, orat different stages of development, or in response to differentenvironmental conditions. It is further recognized that since in mostcases the exact boundaries of regulatory sequences have not beencompletely defined, DNA fragments of some variation may have identicalpromoter activity. Promoters that cause a gene to be expressed in mostcell types at most times are commonly referred to as “constitutivepromoters”.

As used herein, the “3′ non-coding sequences” or “3′ UTR (untranslatedregion) sequence” refer to DNA sequences located downstream of a codingsequence and include polyadenylation recognition sequences and othersequences encoding regulatory signals capable of affecting mRNAprocessing or gene expression. The polyadenylation signal is usuallycharacterized by affecting the addition of polyadenylic acid tracts tothe 3′ end of the mRNA precursor. The use of different 3′ non-codingsequences is exemplified by Ingelbrecht, I. L., et al. (1989) Plant Cell1:671-680.

As used herein, the term “operably linked” refers to the association ofnucleic acid sequences on a single nucleic acid fragment so that thefunction of one is regulated by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of regulatingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in a sense or antisenseorientation. In another example, the complementary RNA regions of theinvention can be operably linked, either directly or indirectly, 5′ tothe target mRNA, or 3′ to the target mRNA, or within the target mRNA, ora first complementary region is 5′ and its complement is 3′ to thetarget mRNA.

As used herein, the term “cross”, “crossing”, “cross pollination” or“cross-breeding” refer to the process by which the pollen of one floweron one plant is applied (artificially or naturally) to the ovule(stigma) of a flower on another plant.

As used herein, the term “gene” refers to any segment of DNA associatedwith a biological function. Thus, genes include, but are not limited to,coding sequences and/or the regulatory sequences required for theirexpression. Genes can also include nonexpressed DNA segments that, forexample, form recognition sequences for other proteins. Genes can beobtained from a variety of sources, including cloning from a source ofinterest or synthesizing from known or predicted sequence information,and may include sequences designed to have desired parameters.

As used herein, the term “vector”, “plasmid”, or “construct” refersbroadly to any plasmid or virus encoding an exogenous nucleic acid. Theterm should also be construed to include non-plasmid and non-viralcompounds which facilitate transfer of nucleic acid into virions orcells, such as, for example, polylysine compounds and the like. Thevector may be a viral vector that is suitable as a delivery vehicle fordelivery of the nucleic acid, or mutant thereof, to a cell, or thevector may be a non-viral vector which is suitable for the same purpose.Examples of viral and non-viral vectors for delivery of DNA to cells andtissues are well known in the art and are described, for example, in Maet al. (1997, Proc. Natl. Acad. Sci. U.S.A. 94:12744-12746). Examples ofviral vectors include, but are not limited to, recombinant plantviruses. Non-limiting examples of plant viruses include, TMV-mediated(transient) transfection into tobacco (Tuipe, T-H et al (1993), J.Virology Meth, 42: 227-239), ssDNA genomes viruses (e.g., familyGeminiviridae), reverse transcribing viruses (e.g., familiesCaulimoviridae, Pseudoviridae, and Metaviridae), dsNRA viruses (e.g.,families Reoviridae and Partitiviridae), (−) ssRNA viruses (e.g.,families Rhabdoviridae and Bunyaviridae), (+) ssRNA viruses (e.g.,families Bromoviridae, Closteroviridae, Comoviridae, Luteoviridae,Potyviridae, Sequiviridae and Tombusviridae) and viroids (e.g., familiesPospiviroldae and Avsunviroidae). Detailed classification information ofplant viruses can be found in Fauquet et al (2008, “Geminivirus straindemarcation and nomenclature”. Archives of Virology 153:783-821,incorporated herein by reference in its entirety), and Khan et al.(Plant viruses as molecular pathogens; Publisher Routledge, 2002, ISBN1560228954, 9781560228950). Examples of non-viral vectors include, butare not limited to, liposomes, polyamine derivatives of DNA, and thelike.

Camelina Sativa

Camelina is a genus of flowering plants belonging to the Brassicaceaefamily. Camelina sativa is a particular species of the genus Camelinathat is important historically and is a source of oil that can be usedin, for example, biofuels and lubricants. C. sativa is beinginvestigated for both biofuel and human utility. It is a crop that hasnot benefited much from molecular investigation in the past and as such,there is relatively little sequence information available. The utilityof a plant oil either for biodiesel or food depends on its fatty acidcomposition. Camelina has a fatty acid composition with high levels ofboth polyunsaturated fatty acids such as 18:2 and 18:3 (52-54%) as wellas long chain fatty acids such as 20:1 (11-15%) and 22:1 (2-5%). Forbiodiesel, the optimum fatty acid is 18:1 (oleic). Oleic has the bestbalance of characteristics for cloud point vs. oxidative stability.Polyunsaturated fatty acids such as 18:2 and 18:3 have poor oxidativestability. The long chain fatty acids such as 20:1 and 22:1 contributeto out of range distillation temperatures in biodiesel. For biodieselutility it is therefore desirable to lower the level of polyunsaturatedfatty acids and to lower the level of long chain fatty acids. Theultimate goal is to increase the percentage of 18:1 fatty acid. 18:1 isalso considered a good fatty acid for food utility.

Camelina has not been intensively bred and the germplasm is somewhatlimited genetically. An in-house field study of a significant number ofcultivars showed little variation in the fatty acid composition. Thisagrees with published literature (e.g., Putnam et al., 1993. Camelina: Apromising low-input oilseed. p. 314-322. In: J. Janick and J. E. Simon(eds.), New crops. Wiley, New York.)

Fatty Acids Synthesis in Plants

Fatty acid biosynthesis in plants takes place within the endoplasmicreticulum and plastids, the latter of which is an organelle widelythought to have originated from a photosynthetic bacterial symbiont.Fatty acid metabolism in plants closely resembles that of bacteria.

During fatty acid biosynthesis, a repeated series of reactionsincorporates acetyl moieties of acetyl-CoA into an acyl group 16 or 18carbons long. The enzymes involved in this synthesis are acetyl-CoAcarboxylase (ACCase), malonyl-CoA:ACP transacylase, 3-ketoacyl-ACPsynthase I and III (KAS I and KAS III), 3-ketoacyl-ACP reductase,2,3-trans-Enoyl-ACP reductase, 3-hydroxyacyl-ACP dehydratase (allreferred as fatty acid synthase (FAS), except for ACCase). The namefatty acid synthase refers to a complex of several individual enzymesthat catalyze the conversion of acetyl-CoA and malonyl-CoA to 16:0 and18:0 fatty acids. Acyl-carrier protein (ACP), an essential proteincofactor, is generally considered a component of FAS.

The last three steps of the fatty acids synthesis cycle reduce a3-ketoacyl substrate to form a fully saturated acyl chain. Each cycle offatty acid synthesis adds two carbons to the acyl chain. Typically,fatty acid synthesis ends at 16:0 or 18:0, when one of several reactionsstops the process. The most common reactions are hydrolysis of acylmoiety from ACP by a thioesterase, transfer of the acyl moiety from ACPdirectly onto a glycerolipid by an acyl transferase, or double-bondformation on the acyl moiety by an acyl-ACP desaturase. The thioesterasereaction yields a sulfhydryl ACP.

Two principal types of acyl-ACP thioesterases occur in plants. Formaking storage lipids (triglycerides) in the ER, the FAT enzymes convertthe fatty acid-ACP to a fatty acid-Co-A. The substrate for FAE1 is anR-CoA and it is an R-CoA that is added to various positions in theglycerol backbone during the Kennedy pathway portion of the synthesis ofTriglycerides in the ER (FIG. 7). The major class, designated FatA, ismost active with 18:1 delta9-ACP. A second class designated FatB,typified by 16:0-ACP thioesterase, is most active with shorter-chain,saturated acyl-ACPs. Thioesterases play important role in plants thathave unusually short fatty acids, such as coconut, many species ofCuphea, and California bay. These plants have thioesterases that areespecially active with C10 to C12 acyl-ACPs, by prematurely terminatingfatty acid biosynthesis.

Unsaturated fatty acids are produced by desaturation of saturated lipidswith the help of desaturases (FAD enzymes). Most fatty acid desaturases(FADs) in plants are integral membrane proteins, with the exception thatplant contains a soluble, plastid-localized stearoyl-ACP desaturase. Thenumber and properties of different FADs in plants are known from theisolation of a comprehensive collection of Arabidopsis mutants withdefects in each of eight desaturase genes. The enzymes encoded by thesegenes differ in substrate specificity, subcellular location, mode ofregulation, or some combination of these. A summary of the ArabidopsisFADs is shown below:

Site of subcellular Fatty acid double-bond Name location substratesinsertion Notes FAD2 ER 18:1Δ9 Δ12 preferred substrate isphosphatidylcholine, oleate desaturase FAD3 ER 18:2Δ9,12 ω3 preferredsubstrate is phosphatidylcholine, linoleate desaturase FAD4 Chloroplast16:0 Δ3 produces 16:1-trans at sn-2 of phosphatidylglycerol FADSChloroplast 16:0 Δ7 desaturates 16:0 at sn-2 ofmonogalactosyldiacylglycerol FAD6 Chloroplast 16:1Δ7 and ω6 acts on allchloroplast glycerolipids, oleate 18:1Δ9 desaturase FAD7 Chloroplast16:247,11 and ω3 acts on all chloroplast glycerolipids, linoleate18:2Δ9,12 desaturase FAD9 Chloroplast 16:247,11 and ω3 isoenzyme of FAD7induced by low temperature, 18:2Δ9,12 linoleate desaturase FAB2Chloroplast 18:0 Δ9 stromal stearoyl-ACP desaturase

The biochemical defect of each class of mutants is shown by breaks inthe pathway on page 480 of Buchanan et al., Biochemistry and MolecularBiology of Plants, American Society of Plant Physiologists, 2000, ISBN0943088372, 9780943088372, which is incorporated by reference in itsentirety.

Extensive surveys of the fatty acid composition of seed oils fromdifferent plant species have resulted in the identification of more than200 naturally occurring fatty acids, which can broadly be classifiedinto 18 structural classes, such as laballenic acid, stearolic acid,sterculynic acid, chaulmoogric acid, ricinoleic acid, vernolic acid,furan-containing fatty acid, et al. Less is known about the mechanismsresponsible for the synthesis and accumulation of unusual fatty acids,or of their significance to the fitness of the plants that accumulatethem. However, recent studies indicate that enzymes involved in thesynthesis of unusual fatty acids are structurally similar to thedesaturases and hydroxylases. Unusual fatty acids occur almostexclusively in seed oils and may serve a defense function.

Synthesis of structural lipids (e.g. cutin, suberin, epicuticular wax)has also been studied in Arabidopsis. Proposed pathways related to thisis shown on page 512 of Buchanan et al., Biochemistry and MolecularBiology of Plants, American Society of Plant Physiologists, 2000, ISBN0943088372, 9780943088372, which is incorporated by reference in itsentirety.

Thus, as used herein, the phrase “fatty acid synthesis genes” or “FASgene” refers to any genes that are involved in synthesis of fatty acids,cuticle, and wax as described above. For example, such genes include,but are not limited to, malonyl-CoA:ACP transacylase, 3-ketoacyl-ACPsynthase I and III (KAS I and KAS III), 3-ketoacyl-ACP reductase,2,3-trans-Enoyl-ACP reductase, 3-hydroxyacyl-ACP dehydratase, acyl-ACPthioesterases, fatty acid desaturases (e.g., FAD2, FADS), fatty acidelongases (e.g., FAE1), hydroxylases, and enzymes displayed in FIGS. 7and 13.

Seed oil of Camelina sativa contains high levels (up to 45%) of omega-3fatty acids, which is uncommon in vegetable sources. Over 50% of thefatty acids in cold pressed Camelina oil are polyunsaturated. The majorcomponents are alpha-linolenic acid—C18:3 (omega-3-fatty acid, approx35-45%) and linoleic acid—C18:2 (omega-6 fatty acid, approx 15-20%).FIG. 11 shows a representative composition of Camelina seed oil. The oilis also very rich in natural antioxidants, such as tocopherols, makingthis highly stable oil very resistant to oxidation and rancidity. It has1-3% erucic acid. The vitamin E content of Camelina oil is approximately110 mg/100 g. The present invention relates to increasing oleic acid(18:1) level, decreasing the level of long chain fatty acids, and/orimproving the seed oil quality of Camelina. As used herein, the term“level” refers to the relative percentage of a component in a mixture.

In the endoplasmic reticulum, oleic acid (18:1) is converted to linoleicacid (18:2) by a delta-12-desaturase, fatty acid desaturase 2 (FAD2).Mutations in Arabidopsis thaliana FAD2 have been shown to increase thelevels of 18:1 in the seeds 2-3.4 fold while decreasing the levels of18:2 fatty acids 4-10 fold. (Levels of 20:1 also increased approximately1.5 fold—Okuley 1994.)

Very long chain fatty acids are synthesized in the cytosol by extensionof an 18 carbon fatty acid. The rate limiting step is thought to be theinitial condensation step, catalyzed in the seed by fatty acid elongase1 (FAE1, Kunst 1992). In Arabidopsis, where approximately 25% of seedfatty acids can be long chain fatty acids, mutants in FAE1 have lessthan 1%. Interestingly, Arabidopsis fae1 mutants show a greater than2-fold increase in 18:1 content in the seeds. (Katavic et al. (2002).“Restoring enzyme activity in nonfunctional low erucic acid Brassicanapus fatty acid elongase 1 by a single amino acid substitution.” Eur JBiochem 269(22): 5625-31.)

FAD2 and FAE1 Genes of Camelina Sativa

The invention discloses the full genomic sequence of three FAD2 genesand three FAE1 genes from Camelina sativa with both upstream anddownstream regions for FAD2 and upstream regions for FAE1 (deposited inGenbank at the NCBI, Genbank IDs: GU929417-GU929422, SEQ ID NOs. 1-6).These sequences include both the coding region as well as severalhundred base pairs upstream and downstream of the genes. The codingsequences for the Camelina sativa FAD2 were obtained using primers fromArabidopsis thaliana FAD2 while the coding regions for the Camelinasativa FAE1 were obtained using primers from Crambe abyssinica FAE1.Also obtained are coding sequences for FAD2 and FAE1 genes from Capsellrubella, A. Lyrata, Camelina hispida, Camelina laxa, Camelinamicrocarpa, and Camelina rumelica (GU929423-GU929441, SEQ ID NOs 45-63),which were amplified using C. Sativa primers. The upstream regions forall the genes were obtained using a combination of RACE PCR and PCR withprimers from upstream Arabidopsis sequences in conjunction with primersto Camelina sequences. The downstream regions of FAD2 were obtainedusing PCR with primers designed from a combination of downstreamArabidopsis sequence in conjunction with primers to Camelina sequences.The Camelina sativa FAD2 and FAE1 genes are highly homologous to bothArabidopsis and Brassica napus (e.g., canola, oilseed rape) FAD2 andFAE1. However, the disclosed sequences are specific to Camelina sativa.

The present invention provides an isolated nucleic acid sequencecomprising a sequence selected from the group consisting of SEQ ID NOs:1 to 6 and SEQ ID NOs: 45-63, and fragments and variations derived fromthereof. In one embodiment, the present invention provides an isolatedpolynucleotide encoding plant fatty acid desaturase, comprising anucleic acid sequence that shares at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, atleast 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least99.7%, at least 99.8%, or at least 99.9% identity to SEQ ID NO: 1, 2, 3,45, 46, 48, 51, 54, 55, 56, 60, and/or 61. In another embodiment, thepresent invention provides an isolated polynucleotide encoding fattyacid elongase, comprising a nucleic acid sequence that shares at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, at least99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%,at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%identity to SEQ ID NO: 4, 5, 6, 47, 49, 50, 52, 53, 57, 58, 59, 62,and/or 63.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman (Adv. Appl. Math., 2:482, 1981); Needleman and Wunsch (J.MoI. Biol., 48:443, 1970); Pearson and Lipman (Proc. Natl. Acad. Sci.,85:2444, 1988); Higgins and Sharp (Gene, 73:237-44, 1988); Higgins andSharp (CABIOS, 5:151-53, 1989); Corpet et al. (Nuc. Acids Res.,16:10881-90, 1988); Huang et al. (Comp. Appls Biosci., 8:155-65, 1992);and Pearson et al. (Meth. Mol. Biol., 24:307-31, 1994). Altschul et al.(Nature Genet., 6:119-29, 1994) presents a detailed consideration ofsequence alignment methods and homology calculations.

The present invention also provides a chimeric gene comprising theisolated nucleic acid sequence of any one of the polynucleotidesdescribed above operably linked to suitable regulatory sequences.

The present invention also provides a recombinant construct comprisingthe chimeric gene as described above. In one embodiment, saidrecombinant construct is a gene silencing construct, such as used inRNAi gene silencing.

The expression vectors of the present invention will preferably includeat least one selectable marker. Such markers include dihydrofolatereductase, G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria. Vectors that can be used with the inventioncomprise vectors for use in bacteria, which comprise pQE70, pQE60 andpQE-9, pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A,pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5. Among preferredeukaryotic vectors are pFastBac1 pWINEO, pSV2CAT, pOG44, pXT1 and pSG,pSVK3, pBPV, pMSG, and pSVL. Other suitable vectors will be readilyapparent to the skilled artisan.

The present invention also provides a transformed host cell comprisingthe chimeric gene as described above. In one embodiment, said host cellis selected from the group consisting of bacteria, yeasts, filamentousfungi, algae, animals, and plants.

These sequences allow the design of gene-specific primers and probes forboth FAD2 and FAE1. Additional data demonstrates that all three copiesof each gene are expressed in the seed, i.e. no one copy is silent inthe seed.

Primers are short nucleic acid molecules, for instance DNAoligonucleotides, usually 7 nucleotides or more in length, for examplethat hybridize to contiguous complementary nucleotides or a sequence tobe amplified. Longer DNA oligonucleotides may be about 15, 20, 25, 30 or50 nucleotides or more in length. Primers can be annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand, and then the primerextended along the target DNA strand by a DNA polymerase enzyme. Primerpairs can be used for amplification of a nucleic acid sequence, forexample, by the PCR or other nucleic-acid amplification methods known inthe art, as described above.

A probe comprises an identifiable, isolated nucleic acid that recognizesa target nucleic acid sequence. A probe includes a nucleic acid that isattached to an addressable location, a detectable label or otherreporter molecule and that hybridizes to a target sequence. Typicallabels include radioactive isotopes, enzyme substrates, co-factors,ligands, chemiluminescent or fluorescent agents, haptens, and enzymes.Methods for labelling and guidance in the choice of labels appropriatefor various purposes are discussed, for example, in Sambrook et al.(ed.), Molecular Cloning: A Laboratory Manual, 2^(nd) ed., vol. 1-3,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989 andAusubel et al. Short Protocols in Molecular Biology, 4^(th) ed., JohnWiley & Sons, Inc., 1999.

Methods for preparing and using nucleic acid probes and primers aredescribed, for example, in Sambrook et al. (ed.), Molecular Cloning: ALaboratory Manual, 2^(nd) ed., vol. 1-3, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989; Ausubel et al. Short Protocols inMolecular Biology, 4^(th) ed., John Wiley & Sons, Inc., 1999; and Inniset al. PCR Protocols, A Guide to Methods and Applications, AcademicPress, Inc., San Diego, Calif., 1990. Amplification primer pairs can bederived from a known sequence, for example, by using computer programsintended for that purpose such as PRIMER (Version 0.5, 1991, WhiteheadInstitute for Biomedical Research, Cambridge, Mass.). One of ordinaryskill in the art will appreciate that the specificity of a particularprobe or primer increases with its length. Thus, in order to obtaingreater specificity, probes and primers can be selected that comprise atleast 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides of atarget nucleotide sequences.

The inventor also obtained Real Time qPCR expression data that showsthat FAD2 and FAE1 genes are expressed in the seed. In addition, SNPexpression data demonstrated that all three copies of FAD2 and of FAE1are expressed. Data that supports these SNP results was also obtainedfrom sequencing a cDNA library from developing Camelina seed.

The invention also provides an EMS mutant library that has been createdin Camelina sativa variety CS32 (commercial name as SO30). InitialTILLING® using primers designed to the three FAD2 genes yielded mutantsin all three FAD2 genes (Hutcheon et al., TILLING® for Altered FattyAcid Profiles in Camelina sativa, July 2009, American Society of PlantBiologists Annual Meeting, which is herein incorporated by reference inits entirety for all purposes). Preliminary analysis on lipidcomposition of these mutants using Gas Chromatography-Flame IonizationDetector (GC-FID) has also been conducted. In addition, Tilling mutantshave been identified in FAE1 and preliminary analysis of lipidcomposition using GC-FID has been conducted on these mutants (Tables19-20).

The close relationship between C. species and the model plantArabidopsis thaliana (Al-Shehbaz, Beilstein et al. 2006; Beilstein,Al-Shehbaz et al. 2006; Beilstein, Al-Shehbaz et al. 2008) facilitatesthe manipulation of known pathways, such as the one regulating fattyacid biosynthesis. C. sativa seed oil is high in both polyunsaturatedand long chain fatty acids (Budin, Breene et al. 1995; Zubr 1997; Gugeland Falk 2006), suggesting that both FAD2 and FAE1 are present andactive. Three copies each of the FAD2 and FAE1 genes were isolated froman agronomic accession of Camelina sativa using primers designed fromArabidopsis thaliana or Crambe abyssinica sequence, Previouslyidentified conserved sites in FAD2 (Tocher D R 1998; McCartney, Dyer etal. 2004; Belo, Zheng et al. 2008) and FAE1 (Ghanevati and Jaworski2001; Moon, Smith et al. 2001; Ghanevati and Jaworski 2002) are presentin all three copies of each gene and a 5′ intron shown to be importantin regulating FAD2 expression in sesame (Kim, Kim et al. 2006) wasidentified in all three CsFAD2 copies. Real Time qPCR data and SequenomMassARRAY SNP analysis of the FAD2 and FAE1 cDNA showed that all threecopies of each gene are expressed in developing seeds. Thus, it seemslikely that all three copies of FAD2 and FAE1 in C. sativa arefunctional.

The cloning of three copies of FAD2 and FAE1 from the C. sativa genome,as well as the observation of three LFY hybridization signals bySouthern analysis could be explained by at least two possible scenarios:segmental duplications of selected regions within a diploid genomeeither through tandem duplications or through transpositions, or wholegenome duplications resulting from polyploidization. The possibilitythat ancient segmental duplications or transpositions affected all threeexamined loci seems less probable than polyploidy. Furthermore, noevidence of recent segmental duplication involving multiple genes hasbeen observed in sequenced plant genomes (Arabidopsis genome (TAIR 2009,2010); rice genome (TIGR Rice Database); maize genome (Maize GenomeBrowser 2010); and Soybean Genome (Phytozome, 2010)).

FAD2 and FAE1 Proteins of Camelina Sativa

The present invention also provides polypeptides and amino acidsequences comprising at least a portion of the isolated protein selectedfrom the group consisting of SEQ ID NOs: 7-12, and all variants thereof.

The present invention also provides an isolated amino acid sequencecomprising a sequence selected from the group consisting of SEQ ID NOs:7 to 12, and fragments and variations derived from thereof. In someembodiments, the present invention provides an isolated polypeptidecomprising an amino acid sequence that shares at least about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or about99.9% identity to SEQ ID NO: 7, 8, 9, 64, 65, 67, 70, 73, 74, 75, 79,and/or 80. In one embodiment, the present invention provides an isolatedpolypeptide comprising an amino acid sequence which encodes an aminoacid sequence that shares at least about 85%, about 86%, about 87%,about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.1%,about 99.2%, about 99.3%, about 99.4%, about 99.5%, about 99.6%, about99.7%, about 99.8%, or about 99.9% identity to SEQ ID NO: 10, 11, 12,66, 68, 69, 71, 72, 76, 77, 78, 81, and/or 82.

The invention also encompasses variants and fragments of proteins ofFAD2 and FAE1 isolated in the present invention. The variants maycontain alterations in the amino acid sequences of the constituentproteins. The term “variant” with respect to a polypeptide refers to anamino acid sequence that is altered by one or more amino acids withrespect to a reference sequence. The variant can have “conservative”changes, or “nonconservative” changes, e.g., analogous minor variationscan also include amino acid deletions or insertions, or both.

Functional fragments and variants of a polypeptide include thosefragments and variants that maintain one or more functions of the parentpolypeptide. It is recognized that the gene or cDNA encoding apolypeptide can be considerably mutated without materially altering oneor more of the polypeptide's functions. First, the genetic code iswell-known to be degenerate, and thus different codons encode the sameamino acids. Second, even where an amino acid substitution isintroduced, the mutation can be conservative and have no material impacton the essential function(s) of a protein. See, e.g., StryerBiochemistry 3rd Ed., 1988. Third, part of a polypeptide chain can bedeleted without impairing or eliminating all of its functions. Fourth,insertions or additions can be made in the polypeptide chain forexample, adding epitope tags, without impairing or eliminating itsfunctions (Ausubel et al. J. Immunol. 159(5): 2502-12, 1997). Othermodifications that can be made without materially impairing one or morefunctions of a polypeptide can include, for example, in vivo or in vitrochemical and biochemical modifications or the incorporation of unusualamino acids. Such modifications include, but are not limited to, forexample, acetylation, carboxylation, phosphorylation, glycosylation,ubiquination, labelling, e.g., with radionucleotides, and variousenzymatic modifications, as will be readily appreciated by those wellskilled in the art. A variety of methods for labelling polypeptides, andlabels useful for such purposes, are well known in the art, and includeradioactive isotopes such as ³²P, ligands which bind to or are bound bylabelled specific binding partners (e.g., antibodies), fluorophores,chemiluminescent agents, enzymes, and anti-ligands. Functional fragmentsand variants can be of varying length. For example, some fragments haveat least 10, 25, 50, 75, 100, 200, or even more amino acid residues.These mutations can be natural or purposely changed. In someembodiments, mutations containing alterations that produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the proteins or how the proteins are made are anembodiment of the invention.

Conservative amino acid substitutions are those substitutions that, whenmade, least interfere with the properties of the original protein, thatis, the structure and especially the function of the protein isconserved and not significantly changed by such substitutions.Conservative substitutions generally maintain (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain. Furtherinformation about conservative substitutions can be found, for instance,in Ben Bassat et al. (J. Bacterial., 169:751-757, 1987), O'Regan et al.(Gene, 77:237-251, 1989), Sahin-Toth et al. (Protein Sci., 3:240-247,1994), Hochuli et al. (Bio/Technology, 6:1321-1325, 1988) and in widelyused textbooks of genetics and molecular biology. The Blosum matricesare commonly used for determining the relatedness of polypeptidesequences. The Blosum matrices were created using a large database oftrusted alignments (the BLOCKS database), in which pairwise sequencealignments related by less than some threshold percentage identity werecounted (Henikoff et al., Proc. Natl. Acad. Sci. USA, 89:10915-10919,1992). A threshold of 90% identity was used for the highly conservedtarget frequencies of the BLOSUM90 matrix. A threshold of 65% identitywas used for the BLOSUM65 matrix. Scores of zero and above in the Blosummatrices are considered “conservative substitutions” at the percentageidentity selected. The following table shows exemplary conservativeamino acid substitutions.

Very Highly- Highly Conserved Original Conserved Substitutions (from theConserved Substitutions Residue Substitutions Blosum90 Matrix) (from theBlosum65 Matrix) Ala Ser Gly, Ser, Thr Cys, Gly, Ser, Thr, Val Arg LysGln, His, Lys Asn, Gln, Glu, His, Lys Asn Gln; His Asp, Gln, His, Lys,Ser, Thr Arg, Asp, Gln, Glu, His, Lys, Ser, Thr Asp Glu Asn, Glu Asn,Gln, Glu, Ser Cys Ser None Ala Gln Asn Arg, Asn, Glu, His, Lys, Met Arg,Asn, Asp, Glu, His, Lys, Met, Ser Glu Asp Asp, Gln, Lys Arg, Asn, Asp,Gln, His, Lys, Ser Gly Pro Ala Ala, Ser His Asn; Gln Arg, Asn, Gln, TyrArg, Asn, Gln, Glu, Tyr Ile Leu; Val Leu, Met, Val Leu, Met, Phe, ValLeu Ile; Val Ile, Met, Phe, Val Ile, Met, Phe, Val Lys Arg; Gln; GluArg, Asn, Gln, Glu Arg, Asn, Gln, Glu, Ser, Met Leu; Ile Gln, Ile, Leu,Val Gln, Ile, Leu, Phe, Val Phe Met; Leu; Tyr Leu, Trp, Tyr Ile, Leu,Met, Trp, Tyr Ser Thr Ala, Asn, Thr Ala, Asn, Asp, Gln, Glu, Gly, Lys,Thr Thr Ser Ala, Asn, Ser Ala, Asn, Ser, Val Trp Tyr Phe, Tyr Phe, TyrTyr Trp; Phe His, Phe, Trp His, Phe, Trp Val Ile; Leu Ile, Leu, Met Ala,Ile, Leu, Met, Thr

In some examples, variants can have no more than 3, 5, 10, 15, 20, 25,30, 40, 50, or 100 conservative amino acid changes (such as very highlyconserved or highly conserved amino acid substitutions). In otherexamples, one or several hydrophobic residues (such as Leu, Ile, Val,Met, Phe, or Trp) in a variant sequence can be replaced with a differenthydrophobic residue (such as Leu, Ile, Val, Met, Phe, or Trp) to createa variant functionally similar to the disclosed FAD2 and FAE1 proteins.

In one embodiment, variants may differ from the disclosed sequences byalteration of the coding region to fit the codon usage bias of theparticular organism into which the molecule is to be introduced. Inother embodiments, the coding region may be altered by taking advantageof the degeneracy of the genetic code to alter the coding sequence suchthat, while the nucleotide sequence is substantially altered, itnevertheless encodes a protein having an amino acid sequencesubstantially similar to the disclosed FAD2 and FAE1 proteins.

Camelina Sativa as an Allohexaploid Plant

The present inventors for the first time in the art demonstrates thatCamelina sativa is an allohexaploid plant.

While not wishing to be bound to any particular theory, triplication ofthe C. sativa genome likely occurred through whole genome duplication,either through autopolyploidization or through allopolyploidization. Anautopolyploidy event might have triplicated a single diploid genomeresulting in an autohexaploid with a haploid genome of 18, 21, or 24chromosomes. Given that C. sativa has a chromosome count of n=20,chromosome splitting or fusion could then have increased the chromosomesfrom 18 to 20, or decreased the chromosomes from 21 or 24 to 20.

Alternatively, triplication of the C. sativa genome might have resultedfrom two allopolyploidy events, resulting in first a tetraploid then ahexaploid, similar to the origin of cultivated wheat. According to thishypothesis, the three copies of each gene diverged in different diploidgenomes before converging through polyploidy events. Taking intoconsideration the reported chromosome counts of various Camelinaspecies, the basal chromosome number of the diploid parental speciescontributing to the C. sativa haploid genome of 20 chromosomes could be7+7+6 or 8+6+6. The allopolyploid hypothesis is supported by theobservation that C. sativa demonstrates diploid inheritance (Gehringer,Friedt et al. 2006; Lu. 2008), as would be expected for an allopolyploid(Sybenga 1996). A hexaploid C. sativa could also be derived from thecombination of an autotetraploid and a diploid species if, in anautopolyploidized genome, homologous chromosomes differentiated so thatthe subsequent chromosome-specific pairing mimicked an allopolyploidgenome in its diploid inheritance patterns. Regardless of itsevolutionary path, the C. sativa genome appears organized in threeredundant and differentiated copies and can be formally considered to bean allohexaploid.

Results from the inventors' phylogenetic analyses support a history ofduplication for both FAD2 and FAE1 in Camelina. For FAD2, duplicationswere only recovered for C. sativa, C. microcarpa, and C. rumelica. Thesedata are consistent with genome size data, which indicate that all threegenomes are larger than C. laxa and C. hispida, from which only a singleFAD2 copy was recovered. Taken together, the results suggest that C.sativa, C. microcarpa, and C. rumelica are likely polyploids. Given theslightly smaller genome size of C. rumelica, and the fact that only twoFAD2 copies were recovered from it, the C. rumelica sampled may betetraploid while C. saliva and C. microcarpa are hexaploid.Interestingly, in both the FAD2 and FAE1 trees, one copy each of C.rumelica and C. microcarpa are strongly supported as sister. Thus, treesfrom these genes indicate that C. rumelica and C. microcarpa are closelyrelated. The various placement of C. microcarpa FAD2 and FAE1 copies canbe explained if C. microcarpa is the result of a hybridization eventbetween C. rumelica and a currently unsampled, and thus unidentifiedspecies of Camelina. Two of the three copies of both FAD2 and FAE1 areidentical, or nearly identical, in C. sativa and C. microcarpa,suggesting that C. sativa and C. microcarpa share a parental genome.Thus, the inventors suggest that an unsampled Camelina speciescontributed its genome to the hybrid formation of both C. saliva and C.microcarpa. In the case of C. microcarpa, the hybridization event likelyinvolved C. rumelica. Given the chromosome count of n=6 for C. rumelica,the other putative parent would be expected to have an x=7 genome, andfurthermore to be tetraploid at n=14. Such a cross would result in theobserved C. microcarpa genome, with chromosome count n=20.Interestingly, C. hispida is the only species we sampled with achromosome count of n=7; however no strong relationship between C.hispida and C. microcarpa is inferred in either gene tree. However, aweak relationship between C. sativa and C. hispida is inferred from theFAE1 tree, and thus the possibility that C. hispida is involved in thepolyploid formation of C. sativa should be explored further.

The likely allohexaploid nature of the Camelina sativa genome hasmultiple implications. Its vigor and adaptability to marginal growthconditions may result at least in part from polyploidy. Polyploids arethought to be more adaptable to new or harsh environments, with theability to expand into broader niches than either progenitor (Brochmann,Brysting et al. 2004; Salmon 2005). Indeed, C. hispida and C. laxa, bothof which are likely diploids, are found only in Turkey, Iran, Armenia,and Azerbaijan, while C. microcarpa and C. sativa are distributedthroughout Asia, Europe, and North Africa and are naturalized in NorthAmerica (GRIN; Akeroyd 1993). The mechanisms behind this increasedadaptability are not completely understood, but have been attributed toheterosis, genetic and regulatory network redundancies, and epigeneticfactors (Comai 2005; Hegarty and Hiscock 2008).

Allohexaploidy might also affect any potential manipulations of the C.sativa genome, such as introgression of geimplasm or induced mutations.Introgression of an exotic germplasm could be facilitated by the type ofpolyploidy-dependent manipulations that are possible in wheat, apotentially comparable allohexaploid (Gill and Friebe 1998; Dubcovskyand Dvorak 2007). In addition, polyploids have displayed excellentresponse to reverse genomics approaches such as Targeting Induced LocalLesions in Genomes (TILLING®) (Slade, Fuerstenberg et al. 2005; Cooper,Till et al. 2008). As in wheat, any recessive induced mutations could bemasked by redundant homologous loci that have maintained function(Stadler 1929; Swaminathan and Rao 1960). This implies that multipleknockout alleles at different homologous sites can be combined toachieve partial or complete suppression of a targeted function(Muramatsu 1963; Li, Huang et al. 2008). We also expect that singlelocus traits, whether transgenic or not, will display diploidinheritance due to preferential intragenomic pairing.

Methods of Altering and/or Improving Camelina Seed Oil Composition

In light of the discovery that Camelina is an allohexaploid plant, thepresent invention provides methods of altering and/or improving Camelinaseed oil composition. As used herein, the term “altering” refers to anychange of fatty acid composition in the seed oil, including but notlimited to compound structure, distribution, relative ratio, and yield,et al. The term “improving” refers to any change in seed oil compositionthat makes the seed oil composition better in one or more qualities forindustrial or nutritional applications. Such improvement includes, butis not limited to, improved quality as meal, improved quality as rawmaterial to produce biofuel, biodiesel, lubricant, more monounsaturatedfatty acids and less polyunsaturated fatty acids, increased stability,lower cloud point, less NOx emissions, reduced trans-fatty acids, et al.

The quality of a biodiesel is greatly dependent upon its composition(Conley S P, Tao B: Biodiesel quality: Is All biodiesel Created Equal?Purdue University Extension; 2006). Polyunsaturated fatty acid methylesters (FAME) have been shown to disproportionately increase oxidationof biodiesel. The temperatures at which biodiesel forms crystals (thecloud point) and at which it can no longer be poured (the pour point)are also affected by composition: saturated FAMEs and long chain FAMEsgreatly increase cloud point and pour point. Biodiesel higher inunsaturated FAMEs are therefore better in colder environments, but havea lower oxidative stability than biodiesel higher in saturated FAMEs.Polyunsaturated FAMEs have also been shown to result in increased NOxemissions while long chain fatty acids result in a biodiesel with toohigh of a distillation temperature by ASTM standards. A biodiesel highin 18:1 and low in polyunsaturated FAMEs and long chain FAMEs is thoughtto be the best compromise, resulting in higher oxidative stability witha low enough cloud point and a high enough cetane number to meetbiodiesel standards (ASTM D6751).

Meal is a significant byproduct of the extraction of the oil fromoilseeds for biofuel. To be able to take advantage of this byproduct asa protein supplement for livestock is essential economically for biofuelproducers. In order for meal from a particular oilseed to be included inlivestock feed in the US, it must be approved by the Association ofAmerican Feed Control Officials (AAFCO). The approval takes into accountfeeding studies in livestock and published studies on the quality of themeal and formulates a definition for the meal that is included in theannually updated AAFCO manual. Currently soybean meal is the best sourcefor animal feed because of its favorable amino acid content and highdigestibility. Another widely used meal comes from Canola, an oilseedrape that has been bred to contain <2% erucic acid (22:1) and <30 μmol/gof glucosinolates. High amounts of erucic acid have been linked to fattydeposits in the heart muscles of animals and glucosinolates lend anunpalatable taste and confer adverse effects on growth in animals.Camelina oil has about 1-4% erucic acid, so the development of lineswith consistently <2% erucic acid is still desirable. Thus theidentification of FAE1 mutants with reduced very long chain fatty acids(VLCFA) such as 22:1 is valuable for the potential to create Camelinavarieties having oil, and thus meal, with <2% erucic acid. Camelina mealhas been tested at least in poultry, goat, cattle (Pilgeram et al.,Camelina sativa, A montana omega-3 and fuel crop, Issues in new cropsand new uses. 2007. J. Janick and A. Whipkey (eds.) and turkeys (Frameet al., Use of Camelina sativa in the Diets of Young Turkeys; J. Appl.Poult. Res. 16:381-386). ASHS Press, Alexandria, Va.). Camelina meal cancurrently be included in the diets of broiler chickens and feedlot beefcattle at no more than 10% (FDA, November 2009). Future feeding studiesmay enable the expansion of Camelina meal to swine, laying hens anddairy cattle.

In one embodiment, the methods relate to increasing monounsaturatedfatty acids (e.g., oleic acids (18:1)) level and/or reducingpolyunsaturated fatty acids level in the seed oil, wherein the methodcomprises disrupting and/or altering one or more copies of one or moreCamelina fatty acids synthesis genes. In some embodiments, one, two, orall three copies of Camelina FAD2 and/or FAE1 genes are disrupted. Forexample, the methods comprise utilizing one or more Camelina mutants inany one of the mutations listed in Tables 7 to 12 described in Example11.

In some embodiments, the methods related to increasing monounsaturatedfatty acids (e.g., oleic acids (18:1)) level and/or decreasing very longchain fatty acids (>18 carbons), wherein the methods comprise disruptingand/or altering one or more copies of two or more Camelina fatty acidssynthesis genes. In some embodiments, one, two, or all three copies ofCamelina FAD2 and one, two, or all three FAE1 genes are disrupted.

In some embodiments, mutations in one or more copies of FAD2 genesand/or one or more copies of FAE1 genes described in the Tables 7 to 12are integrated together to create mutant plants with double, triple,quadruple et al. mutations. Such mutants can be created by classicbreeding methods.

In some embodiments, mutations described in the Tables 7-12 can beintegrated into Camelina cultivars other than Cs32 by classic breedingmethods, with or without the help of marker-facilitated gene transfermethods.

In some embodiments, mutations described in the Tables 7-12 can beintegrated into species closely related to Camelina sativa, such asother species in the Brassicaceae family, such as Brassica oleracea(cabbage, cauliflower, etc.), Brassica rapa (turnip, Chinese cabbage,etc.), Brassica napus (rapeseed, etc.), Raphanus sativus (commonradish), Armoracia rusticana (horseradish), Matthiola (stock), and manyothers, with or without the help of marker-facilitated inter-cultivargene transfer methods.

In one embodiment, mutants in Tables 7 to 12, wherein the mutants are inevolutionarily conserved regions or sites can be used to produceCamelina plants with improved or altered seed oil. In one embodiment,mutants in Table 7 to 12, wherein the mutant is due to nonsense mutation(premature stop codon), can be used to produce Camelina plants withimproved or altered seed oil.

In one embodiment, mutants in Tables 7 to 12, wherein the mutants arenot in evolutionarily conserved regions or sites, can also be used toproduce Camelina plants with improved or altered seed oil. Non-limitingexamples of improved seed oil are those having increased oleic acid,increased fatty acids of C18 or less (C≦18), decreased very long chainfatty acid (C>18), and/or decreased polyunsaturated fatty acids, inratio and/or in absolute weight. As used herein, the term “C≦18” refersto a chemical compound having not more than 18 carbons; as used herein,the term C>18 refers to a chemical compound that has more than 18carbons.

In other embodiments, amino acids in conserved domains or sites ofCamelina FAD2 or FAE1 proteins can be compared to FAD2 or FAE1 orthologsin other species, e.g., closely related Brassicaceae species, or plantspecies with known FAD/FAE sequences, which do not contain mutationslisted in Tables 7 to 12. Then, the FAD/FAE genes in these relatedspecies can be substituted or deleted to make mutants with reduced orabolished activity.

In one embodiment, the oleic acid level in the seed oil produced fromthe Camelina plants of the present invention is increased as compared tothe same plants known in the prior art (e.g., comparable wild typeplant). For example, the level of oleic acid in the seed oil isincreased by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%,about 7%, about 8%, about 9%, about 10%, about 12%, about 14%, about16%, about 18%, about 19%, about 20%, about 22%, about 24%, about 26%,about 28%, about 30%, about 32%, about 34%, about 36%, about 38%, about40%, about 42%, about 44%, about 46%, about 48%, about 50%, about 52%,about 54%, about 56%, about 58%, about 59%, about 60%, about 62%, about64%, about 66%, about 68%, about 70%, about 75%, about 80%, about 85%,about 90%, about 95%, about 100%, about 150%, about 200%, about 250%,about 300%, about 350%, about 400%, about 450%, or about 500%.

In another embodiment, the oleic acid yield in the seed oil produced perCamelina plant of the present invention is increased as compared to thesame plants known in the prior art (e.g., comparable wild type plant).As used herein, the term “yield” refers to amount of one or more typesof fatty acids produced per plant, or per acre. For example, the yieldof oleic acid in the seed oil is increased by about 1%, about 2%, about3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about10%, about 12%, about 14%, about 16%, about 18%, about 19%, about 20%,about 22%, about 24%, about 26%, about 28%, about 30%, about 32%, about34%, about 36%, about 38%, about 40%, about 42%, about 44%, about 46%,about 48%, about 50%, about 52%, about 54%, about 56%, about 58%, about59%, about 60%, about 62%, about 64%, about 66%, about 68%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about150%, about 200%, about 250%, about 300%, about 350%, about 400%, about450%, or about 500%.

In another embodiment, the polyunsaturated fatty acid level and/or yieldin the seed oil produced from the Camelina plants of the presentinvention is decreased as compared to the same plants known in the priorart (e.g., comparable wild type plant). For example, the level and/oryield of polyunsaturated fatty acid in the seed oil is decreased byabout 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%,about 8%, about 9%, about 10%, about 12%, about 14%, about 16%, about18%, about 19%, about 20%, about 22%, about 24%, about 26%, about 28%,about 30%, about 32%, about 34%, about 36%, about 38%, about 40%, about42%, about 44%, about 46%, about 48%, about 50%, about 52%, about 54%,about 56%, about 58%, about 59%, about 60%, about 62%, about 64%, about66%, about 68%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, about 100%, about 150%, about 200%, about 250%, about 300%,about 350%, about 400%, about 450%, or about 500%.

In another embodiment, the very long chain fatty acid (C>18) leveland/or yield in the seed oil produced from the Camelina plants of thepresent invention is decreased as compared to the same plants known inthe prior art (e.g., comparable wild type plant). For example, the leveland/or yield of very long chain fatty acid in the seed oil is decreasedby about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%,about 8%, about 9%, about 10%, about 12%, about 14%, about 16%, about18%, about 19%, about 20%, about 22%, about 24%, about 26%, about 28%,about 30%, about 32%, about 34%, about 36%, about 38%, about 40%, about42%, about 44%, about 46%, about 48%, about 50%, about 52%, about 54%,about 56%, about 58%, about 59%, about 60%, about 62%, about 64%, about66%, about 68%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, about 100%, about 150%, about 200%, about 250%, about 300%,about 350%, about 400%, about 450%, or about 500%.

In another embodiment, the fatty acids of C18 or less level and/or yieldin the seed oil produced from the Camelina plants of the presentinvention is increased as compared to the same plants known in the priorart (e.g., comparable wild type plant). For example, the level and/oryield of fatty acids of C18 or less in the seed oil is increased byabout 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%,about 8%, about 9%, about 10%, about 12%, about 14%, about 16%, about18%, about 19%, about 20%, about 22%, about 24%, about 26%, about 28%,about 30%, about 32%, about 34%, about 36%, about 38%, about 40%, about42%, about 44%, about 46%, about 48%, about 50%, about 52%, about 54%,about 56%, about 58%, about 59%, about 60%, about 62%, about 64%, about66%, about 68%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, about 100%, about 150%, about 200%, about 250%, about 300%,about 350%, or about 400%.

Molecular markers are used for the visualization of differences innucleic acid sequences. This visualization is possible due to DNA-DNAhybridization techniques (RFLP) and/or due to techniques using thepolymerase chain reaction (e.g. STS, microsatellites, AFLP, SNP, IMP etal.). All differences between two parental genotypes will segregate in amapping population based on the cross of these parental genotypes. Thesegregation of the different markers may be compared and recombinationfrequencies can be calculated. The recombination frequencies ofmolecular markers on different chromosomes is generally 50%. Betweenmolecular markers located on the same chromosome the recombinationfrequency depends on the distance between the markers. A lowrecombination frequency corresponds to a low distance between markers ona chromosome. Comparing all recombination frequencies will result in themost logical order of the molecular markers on the chromosomes. Thismost logical order can be depicted in a linkage map.

Molecular markers for the present invention, for example, can begenerated by analyzing progeny of a cross between e.g., Cs32 cultivar toanother Camelina species, e.g., Camelina microcarpa. The presentinventors have generated such progeny and more Inter MITE Polymorphisms(IMP) markers can be generated following the procedures outlined in thepresent application. IMP markers are developed by and exclusive to DNALandMarks Inc. IMP markers are based on Miniature Inverted-repeatTransposable Elements (MITEs), which are short interspersed DNAtransposons with terminal inverted repeats (TIRs). They are small insize (<500 bp), conserved TIRs, high A+T content, and consist of severaldistinct families such as Tourist-like, Stowaway-like. They present inplants, fungi, vertebrates, fishes, insects. In plants, they are highlyassociated with genes (flanking regions, introns). They are alsoabundant in plants (several thousand copies per genome). IMP markershave many unique advantages:

Naturally multiplexed—Greatly lowers cost/data point

Reliable—PCR based, reproducible results

Portable—Markers are cross-applicable in all crops

Practical—Useful in a variety of marker-assisted breeding functions

Similarly, Cs32 can be crossed to other species in the Brassicaceaefamily to generate molecular markers for further applications.

In some other embodiments, one, two, or all three copies of CamelinaFAD2 and/or FAE1 genes, and one, two, or all three copies of othernon-FAD2, non-FAE1 fatty acid synthesis genes are disrupted. As usedherein, the phrase “non-FAD, non-FAE fatty acid synthesis genes” refersto polynucleotides encoding polypeptides that are involved in plantfatty acid synthesis, but share less than 95% identity to FAD2 or FAE1polypeptide disclosed in the present invention. In still someembodiments, one, two, or all three copies of Camelina FAD2 and/or FAE1genes are disrupted, while one or more non-FAD, non-FAE fatty acidsynthesis genes are overexpressed. In still more embodiments, one, two,or all three copies of Camelina FAD2 and/or FAE1 genes are disrupted,while one or more non-fatty-acid-synthesis genes are overexpressedand/or disrupted. As used herein, the phrase “non-fatty-acid-synthesisgenes” refers to polynucleotides encoding polypeptides that are notdirectly involved in the synthesis of fatty acids.

According to the present invention, one skilled in the art will be ableto pick preferred target genes and decide when disruption oroverexpression is needed to achieve certain goals, e.g., an induction orreduction of certain fatty acids composition, based on the plant fattyacid metabolic pathways and metabolic analysis tools known in the art(e.g., MetaCyc and AraCyc database, see Zhang et al., Plant Physiology,2005, 138:27-37). For example, one skilled in the art would be able tocombine FAD2 and/or FAE1 loss-of-function mutants (e.g., mutants withreduced, or abolished FAD2 and/or FAE1 protein activity), FAD2 and/orFAE1 gain-of-function mutants (e.g., mutants with altered or increasedFAD2 and/or FAE1 protein activity), or FAD2 and/or FAE1 overexpressionwith overexpression or disruption of non-FAD, non-FAE fatty acid genesto modulate the fatty acid synthesis in a plant. While not wishing to bebound by any particular theory, knock-down of FAD2 can potentially lower18:2 fatty acid; knock-down of FAD3 can potentially lower 18:3 fattyacid; overexpressing plastidial enzyme Δ9 will give higher 18:1;knock-down of both FAD2 and FAD3 will contribute to a higher cloud pointof the oil; knock-down of thioesterases (e.g., FAT A and/or FAT B) willlower the amount of 16:0 fatty acids; knock-down of fatty acid elongase(FAE) will lower the amount of long-chain fatty acids; a dominantnegative KRP protein or a REV protein can increase cell size and thusincrease oil production (see US 2008/263727 and US 2007/056058,incorporated by reference in their entireties).

In addition, using the compositions and methods of the presentinvention, one skilled in the art will be able to combine disruption ofFAD2 and/or FAE1 genes with other mutants and/or transgenes which cangenerally improve plant health, plant biomass, plant resistance tobiotic and abiotic factors, plant yields, wherein the final preferredfatty acid production is increased. Such mutants and/or transgenesinclude, but are not limited to, cell cycle controlling genes, cell sizecontrolling genes, cell division controlling genes, pathogen resistancegenes, and genes controlling plant traits related to seed yield, whichare well known to one skilled in the art (e.g., REV genes, KRP genes).

Methods of disrupting and/or altering a target gene have been known toone skilled in the art. These methods include, but are not limited to,mutagenesis (e.g., chemical mutagenesis, radiation mutagenesis,transposon mutagenesis, insertional mutagenesis, signature taggedmutagenesis, site-directed mutagenesis, and natural mutagenesis),knock-outs/knock-ins, antisense and RNA interference. Various types ofmutagenesis can be used to produce and/or isolate variant nucleic acidsthat encode for protein molecules and/or to further modify/mutate theproteins of the present invention. They include but are not limited tosite-directed, random point mutagenesis, homologous recombination (DNAshuffling), mutagenesis using uracil containing templates,oligonucleotide-directed mutagenesis, phosphorothioate-modified DNAmutagenesis, mutagenesis using gapped duplex DNA or the like. Additionalsuitable methods include point mismatch repair, mutagenesis usingrepair-deficient host strains, restriction-selection andrestriction-purification, deletion mutagenesis, mutagenesis by totalgene synthesis, double-strand break repair, and the like. Mutagenesis,e.g., involving chimeric constructs, is also included in the presentinvention. In one embodiment, mutagenesis can be guided by knowninformation of the naturally occurring molecule or altered or mutatednaturally occurring molecule, e.g., sequence, sequence comparisons,physical properties, crystal structure or the like. For more informationof mutagenesis in plants, such as agents, protocols, see Acquaah et al.(Principles of plant genetics and breeding, Wiley-Blackwell, 2007, ISBN1405136464, 9781405136464, which is herein incorporated by reference inits entity). Methods of disrupting plant genes using RNA interference isdescribed later in the specification.

The present invention provides methods of producing Camelina seed oilcontaining altered and/or increased levels of oleic acid (18:1), and/oraltered or reduced levels of polyunsaturated fatty acids, and/ordecreased very long chain fatty acids (C>18). Such methods compriseutilizing the Camelina plants comprising the chimeric genes as describedabove, or Camelina plants with disrupted FAD2 and/or FAE1 genes asdescribed above.

The present invention also provides methods of breeding Camelina speciesproducing altered levels of fatty acids in the seed oil and/or meal. Inone embodiment, such methods comprise

i) making a cross between the Camelina mutants with mutations asdescribed above to a second Camelina species to make F1 plants;ii) backcrossing said F1 plants to said second Camelina species;iii) repeating backcrossing step until said mutations are integratedinto the genome of said second Camelina species. Optionally, such methodcan be facilitated by molecular markers.

The present invention provides methods of breeding species close toCamelina sativa, wherein said species produces altered levels of fattyacids in the seed oil and/or meal. In one embodiment, such methodscomprise

i) making a cross between the Camelina mutants with mutations asdescribed above to a species close to Camelina sativa to make F1 plants;ii) backcrossing said F1 plants to said species that is close toCamelina sativa;iii) repeating backcrossing step until said mutations are integratedinto the genome of said species that is close to Camelina sativa.Special techniques (e.g., somatic hybridization) may be necessary inorder to successfully transfer a gene from Camelina sativa to anotherspecies and/or genus, such as to B. oleracea. Optionally, such methodcan be facilitated by molecular markers.

Plant Transformation

The present polynucleotides of the present invention can be transformedinto a Camelina plant, or other plants.

The most common method for the introduction of new genetic material intoa plant genome involves the use of living cells of the bacterialpathogen Agrobacterium tumefaciens to literally inject a piece of DNA,called transfer or T-DNA, into individual plant cells (usually followingwounding of the tissue) where it is targeted to the plant nucleus forchromosomal integration. There are numerous patents governingAgrobacterium mediated transformation and particular DNA deliveryplasmids designed specifically for use with Agrobacterium—for example,U.S. Pat. No. 4,536,475, EP0265556, EP0270822, WO8504899, WO8603516,U.S. Pat. No. 5,591,616, EP0604662, EP0672752, WO8603776, WO9209696,WO9419930, WO9967357, U.S. Pat. No. 4,399,216, WO8303259, U.S. Pat. No.5,731,179, EP068730, WO9516031, U.S. Pat. No. 5,693,512, U.S. Pat. No.6,051,757 and EP904362A1. Agrobacterium-mediated plant transformationinvolves as a first step the placement of DNA fragments cloned onplasmids into living Agrobacterium cells, which are then subsequentlyused for transformation into individual plant cells.Agrobacterium-mediated plant transformation is thus an indirect planttransformation method. Methods of Agrobacterium-mediated planttransformation that involve using vectors with no T-DNA are also wellknown to those skilled in the art and can have applicability in thepresent invention. See, for example, U.S. Pat. No. 7,250,554, whichutilizes P-DNA instead of T-DNA in the transformation vector.

Direct plant transformation methods using DNA have also been reported.The first of these to be reported historically is electroporation, whichutilizes an electrical current applied to a solution containing plantcells (M. E. Fromm et al., Nature, 319, 791 (1986); H. Jones et al.,Plant Mol. Biol., 13, 501 (1989) and H. Yang et al., Plant Cell Reports,7, 421 (1988). Another direct method, called “biolistic bombardment”,uses ultrafine particles, usually tungsten or gold, that are coated withDNA and then sprayed onto the surface of a plant tissue with sufficientforce to cause the particles to penetrate plant cells, including thethick cell wall, membrane and nuclear envelope, but without killing atleast some of them (U.S. Pat. No. 5,204,253, U.S. Pat. No. 5,015,580). Athird direct method uses fibrous forms of metal or ceramic consisting ofsharp, porous or hollow needle-like projections that literally impalethe cells, and also the nuclear envelope of cells. Both silicon carbideand aluminium borate whiskers have been used for plant transformation(Mizuno et al., 2004; Petolino et al., 2000; U.S. Pat. No. 5,302,523 USApplication 20040197909) and also for bacterial and animaltransformation (Kaepler et al., 1992; Raloff, 1990; Wang, 1995). Thereare other methods reported, and undoubtedly, additional methods will bedeveloped. However, the efficiencies of each of these indirect or directmethods in introducing foreign DNA into plant cells are invariablyextremely low, making it necessary to use some method for selection ofonly those cells that have been transformed, and further, allowinggrowth and regeneration into plants of only those cells that have beentransformed.

For efficient plant transformation, a selection method must be employedsuch that whole plants are regenerated from a single transformed celland every cell of the transformed plant carries the DNA of interest.These methods can employ positive selection, whereby a foreign gene issupplied to a plant cell that allows it to utilize a substrate presentin the medium that it otherwise could not use, such as mannose or xylose(for example, refer U.S. Pat. No. 5,767,378; U.S. Pat. No. 5,994,629).More typically, however, negative selection is used because it is moreefficient, utilizing selective agents such as herbicides or antibioticsthat either kill or inhibit the growth of nontransformed plant cells andreducing the possibility of chimeras. Resistance genes that areeffective against negative selective agents are provided on theintroduced foreign DNA used for the plant transformation. For example,one of the most popular selective agents used is the antibiotickanamycin, together with the resistance gene neomycin phosphotransferase(nptII), which confers resistance to kanamycin and related antibiotics(see, for example, Messing & Vierra, Gene 19: 259-268 (1982); Bevan etal., Nature 304:184-187 (1983)). However, many different antibiotics andantibiotic resistance genes can be used for transformation purposes(refer U.S. Pat. No. 5,034,322, U.S. Pat. No. 6,174,724 and U.S. Pat.No. 6,255,560). In addition, several herbicides and herbicide resistancegenes have been used for transformation purposes, including the bargene, which confers resistance to the herbicide phosphinothricin (Whiteet al., Nuel Acids Res 18: 1062 (1990), Spencer et al., Theor Appl Genet79: 625-631(1990), U.S. Pat. No. 4,795,855, U.S. Pat. No. 5,378,824 andU.S. Pat. No. 6,107,549). In addition, the dhfr gene, which confersresistance to the anticancer agent methotrexate, has been used forselection (Bourouis et al., EMBO J. 2(7): 1099-1104 (1983).

The expression control elements used to regulate the expression of agiven protein can either be the expression control element that isnormally found associated with the coding sequence (homologousexpression element) or can be a heterologous expression control element.A variety of homologous and heterologous expression control elements areknown in the art and can readily be used to make expression units foruse in the present invention. Transcription initiation regions, forexample, can include any of the various opine initiation regions, suchas octopine, mannopine, nopaline and the like that are found in the Tiplasmids of Agrobacterium tumefaciens. Alternatively, plant viralpromoters can also be used, such as the cauliflower mosaic virus 19S and35S promoters (CaMV 19S and CaMV 35S promoters, respectively) to controlgene expression in a plant (U.S. Pat. Nos. 5,352,605; 5,530,196 and5,858,742 for example). Enhancer sequences derived from the CaMV canalso be utilized (U.S. Pat. Nos. 5,164,316; 5,196,525; 5,322,938;5,530,196; 5,352,605; 5,359,142; and 5,858,742 for example). Lastly,plant promoters such as prolifera promoter, fruit specific promoters,Ap3 promoter, heat shock promoters, seed specific promoters, etc. canalso be used.

Either a gamete-specific promoter, a constitutive promoter (such as theCaMV or Nos promoter), an organ-specific promoter (such as the E8promoter from tomato), or an inducible promoter is typically ligated tothe protein or antisense encoding region using standard techniques knownin the art. The expression unit may be further optimized by employingsupplemental elements such as transcription terminators and/or enhancerelements. For example, the 5′ introns of FAD2 gene in sesame have beendemonstrated to increase and/or regulate expression of certain genes(Kim et al. 2006. Mol Genet Genomics 276(4): 351-68). Thus, the 5′intron sequences of the FAD2 genes of the present invention can be usedto increase expression of either a FAD2 or a non-FAD2 gene. Theexpression cassette can comprise, for example, a seed-specific promoter(e.g. the phaseolin promoter (U.S. Pat. No. 5,504,200). The term“seed-specific promoter”, means that a gene expressed under the controlof the promoter is predominantly expressed in plant seeds with no or nosubstantial expression, typically less than 10% of the overallexpression level, in other plant tissues. Seed specific promoters havebeen well known in the art, for example, U.S. Pat. Nos. 5,623,067,5,717,129, 6,403,371, 6,566,584, 6,642,437, 6,777,591, 7,081,565,7,157,629, 7,192,774, 7,405,345, 7,554,006, 7,589,252, 7,595,384,7,619,135, 7,642,346, and US Application Publication Nos. 20030005485,20030172403, 20040088754, 20040255350, 20050125861, 20050229273,20060191044, 20070022502, 20070118933, 20070199098, 20080313771, and20090100551.

Thus, for expression in plants, the expression units will typicallycontain, in addition to the protein sequence, a plant promoter region, atranscription initiation site and a transcription termination sequence.Unique restriction enzyme sites at the 5′ and 3′ ends of the expressionunit are typically included to allow for easy insertion into apre-existing vector.

In the construction of heterologous promoter/structural gene orantisense combinations, the promoter is preferably positioned about thesame distance from the heterologous transcription start site as it isfrom the transcription start site in its natural setting. As is known inthe art, however, some variation in this distance can be accommodatedwithout loss of promoter function.

In addition to a promoter sequence, the expression cassette can alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes. If the mRNA encoded by the structural gene is tobe efficiently processed, DNA sequences which direct polyadenylation ofthe RNA are also commonly added to the vector construct. Polyadenylationsequences include, but are not limited to the Agrobacterium octopinesynthase signal (Gielen et al., EMBO J 3:835-846 (1984)) or the nopalinesynthase signal (Depicker et al., Mol. and Appl. Genet. 1:561-573(1982)). The resulting expression unit is ligated into or otherwiseconstructed to be included in a vector that is appropriate for higherplant transformation. One or more expression units may be included inthe same vector. The vector will typically contain a selectable markergene expression unit by which transformed plant cells can be identifiedin culture. Usually, the marker gene will encode resistance to anantibiotic, such as G418, hygromycin, bleomycin, kanamycin, orgentamicin or to an herbicide, such as glyphosate (Round-Up) orglufosinate (BASTA) or atrazine. Replication sequences, of bacterial orviral origin, are generally also included to allow the vector to becloned in a bacterial or phage host; preferably a broad host range forprokaryotic origin of replication is included. A selectable marker forbacteria may also be included to allow selection of bacterial cellsbearing the desired construct. Suitable prokaryotic selectable markersinclude resistance to antibiotics such as ampicillin, kanamycin ortetracycline. Other DNA sequences encoding additional functions may alsobe present in the vector, as is known in the art. For instance, in thecase of Agrobacterium transformations, T-DNA sequences will also beincluded for subsequent transfer to plant chromosomes.

To introduce a desired gene or set of genes by conventional methodsrequires a sexual cross between two lines, and then repeatedback-crossing between hybrid offspring and one of the parents until aplant with the desired characteristics is obtained. This process,however, is restricted to plants that can sexually hybridize, and genesin addition to the desired gene will be transferred.

Recombinant DNA techniques allow plant researchers to circumvent theselimitations by enabling plant geneticists to identify and clone specificgenes for desirable traits, such as improved fatty acid composition, andto introduce these genes into already useful varieties of plants. Oncethe foreign genes have been introduced into a plant, that plant can thenbe used in conventional plant breeding schemes (e.g., pedigree breeding,single-seed-descent breeding schemes, reciprocal recurrent selection) toproduce progeny which also contain the gene of interest.

Genes can be introduced in a site directed fashion using homologousrecombination. Homologous recombination permits site-specificmodifications in endogenous genes and thus inherited or acquiredmutations may be corrected, and/or novel alterations may be engineeredinto the genome. Homologous recombination and site-directed integrationin plants are discussed in, for example, U.S. Pat. Nos. 5,451,513;5,501,967 and 5,527,695.

Methods of producing transgenic plants are well known to those ofordinary skill in the art. Transgenic plants can now be produced by avariety of different transformation methods including, but not limitedto, electroporation; microinjection; microprojectile bombardment, alsoknown as particle acceleration or biolistic bombardment; viral-mediatedtransformation; and Agrobacterium-mediated transformation. See, forexample, U.S. Pat. Nos. 5,405,765; 5,472,869; 5,538,877; 5,538,880;5,550,318; 5,641,664; 5,736,369 and 5,736369; International PatentApplication Publication Nos. WO2002/038779 and WO/2009/117555; Lu etal., (Plant Cell Reports, 2008, 27:273-278); Watson et al., RecombinantDNA, Scientific American Books (1992); Hinchee et al., Bio/Tech.6:915-922 (1988); McCabe et al., Bio/Tech. 6:923-926 (1988); Toriyama etal., Bio/Tech. 6:1072-1074 (1988); Fromm et al., Bio/Tech. 8:833-839(1990); Mullins et al., Bio/Tech. 8:833-839 (1990); Hiei et al., PlantMolecular Biology 35:205-218 (1997); Ishida et al., Nature Biotechnology14:745-750 (1996); Zhang et al., Molecular Biotechnology 8:223-231(1997); Ku et al., Nature Biotechnology 17:76-80 (1999); and, Raineri etal., Bio/Tech. 8:33-38 (1990)), each of which is expressly incorporatedherein by reference in their entirety.

Agrobacterium tumefaciens is a naturally occurring bacterium that iscapable of inserting its DNA (genetic information) into plants,resulting in a type of injury to the plant known as crown gall. Mostspecies of plants can now be transformed using this method, includingcucurbitaceous species.

Microprojectile bombardment is also known as particle acceleration,biolistic bombardment, and the gene gun (Biolistic® Gene Gun). The genegun is used to shoot pellets that are coated with genes (e.g., fordesired traits) into plant seeds or plant tissues in order to get theplant cells to then express the new genes. The gene gun uses an actualexplosive (.22 caliber blank) to propel the material. Compressed air orsteam may also be used as the propellant. The Biolistic® Gene Gun wasinvented in 1983-1984 at Cornell University by John Sanford, EdwardWolf, and Nelson Allen. It and its registered trademark are now owned byE. I. du Pont de Nemours and Company. Most species of plants have beentransformed using this method.

A transgenic plant formed using Agrobacterium transformation methodstypically contains a single gene on one chromosome, although multiplecopies are possible. Such transgenic plants can be referred to as beinghemizygous for the added gene. A more accurate name for such a plant isan independent segregant, because each transformed plant represents aunique T-DNA integration event (U.S. Pat. No. 6,156,953). A transgenelocus is generally characterized by the presence and/or absence of thetransgene. A heterozygous genotype in which one allele corresponds tothe absence of the transgene is also designated hemizygous (U.S. Pat.No. 6,008,437).

Breeding Methods

Classic breeding methods can be included in the present invention tointroduce one or more mutations of the present invention into otherCamelina varieties, or other close-related species of the Brassicaceaefamily that are compatible to be crossed with Camelina. In oneembodiment, the mutations are on the FAD2 A, FAD2 B, and/or FAD2 Cgenes. In one embodiment, the mutations are on the FAE1 A, FAE1 B,and/or FAE1 C genes. In one embodiment, the mutations are on any FAD2gene and/or any FAE1 gene.

Open-Pollinated Populations.

The improvement of open-pollinated populations of such crops as rye,many maizes and sugar beets, herbage grasses, legumes such as alfalfaand clover, and tropical tree crops such as cacao, coconuts, oil palmand some rubber, depends essentially upon changing gene-frequenciestowards fixation of favorable alleles while maintaining a high (but farfrom maximal) degree of heterozygosity. Uniformity in such populationsis impossible and trueness-to-type in an open-pollinated variety is astatistical feature of the population as a whole, not a characteristicof individual plants. Thus, the heterogeneity of open-pollinatedpopulations contrasts with the homogeneity (or virtually so) of inbredlines, clones and hybrids.

Population improvement methods fall naturally into two groups, thosebased on purely phenotypic selection, normally called mass selection,and those based on selection with progeny testing. Interpopulationimprovement utilizes the concept of open breeding populations; allowinggenes to flow from one population to another. Plants in one population(cultivar, strain, ecotype, or any germplasm source) are crossed eithernaturally (e.g., by wind) or by hand or by bees (commonly Apis melliferaL. or Megachile rotundata F.) with plants from other populations.Selection is applied to improve one (or sometimes both) population(s) byisolating plants with desirable traits from both sources.

There are several primary methods of open-pollinated populationimprovement. First, there is the situation in which a population ischanged en masse by a chosen selection procedure. The outcome is animproved population that is indefinitely propagable by random-matingwithin itself in isolation. Second, the synthetic variety attains thesame end result as population improvement but is not itself propagableas such; it has to be reconstructed from parental lines or clones.Third, a method used in plant species that are largely self pollinatedin nature, such as soybeans, wheat, rice, safflower, camelina and othersis pedigree selection. In this situation, crosses are made andindividual plants and lines from individual plants are selected fordesired traits. These lines are thn advanced as genetically homogeneousvarieties. Since the individuals are largely self pollinated these linesare analogous to an inbred line with favorable agronomiccharacteristics. These plant breeding procedures for improvingopen-pollinated populations are well known to those skilled in the artand comprehensive reviews of breeding procedures routinely used forimproving cross-pollinated plants are provided in numerous texts andarticles, including: Allard, Principles of Plant Breeding, John Wiley &Sons, Inc. (1960); Simmonds, Principles of Crop Improvement, LongmanGroup Limited (1979); Hallauer and Miranda, Quantitative Genetics inMaize Breeding, Iowa State University Press (1981); and, Jensen, PlantBreeding Methodology, John Wiley & Sons, Inc. (1988).

Mass Selection.

In mass selection, desirable individual plants are chosen, harvested,and the seed composited without progeny testing to produce the followinggeneration. Since selection is based on the maternal parent only, andthere is no control over pollination, mass selection amounts to a formof random mating with selection. As stated above, the purpose of massselection is to increase the proportion of superior genotypes in thepopulation.

Synthetics.

A synthetic variety is produced by crossing inter se a number ofgenotypes selected for good combining ability in all possible hybridcombinations, with subsequent maintenance of the variety by openpollination. Whether parents are (more or less inbred) seed-propagatedlines, as in some sugar beet and beans (Vicia) or clones, as in herbagegrasses, clovers and alfalfa, makes no difference in principle. Parentsare selected on general combining ability, sometimes by test crosses ortoperosses, more generally by polycrosses. Parental seed lines may bedeliberately inbred (e.g. by selfing or sib crossing). However, even ifthe parents are not deliberately inbred, selection within lines duringline maintenance will ensure that some inbreeding occurs. Clonal parentswill, of course, remain unchanged and highly heterozygous.

Whether a synthetic can go straight from the parental seed productionplot to the farmer or must first undergo one or two cycles ofmultiplication depends on seed production and the scale of demand forseed. In practice, grasses and clovers are generally multiplied once ortwice and are thus considerably removed from the original synthetic.

While mass selection is sometimes used, progeny testing is generallypreferred for polycrosses, because of their operational simplicity andobvious relevance to the objective, namely exploitation of generalcombining ability in a synthetic.

The number of parental lines or clones that enter a synthetic varywidely. In practice, numbers of parental lines range from 10 to severalhundred, with 100-200 being the average. Broad based synthetics formedfrom 100 or more clones would be expected to be more stable during seedmultiplication than narrow based synthetics.

Pedigreed Varieties.

A pedigreed variety is a superior genotype developed from selection ofindividual plants out of a segregating population followed bypropagation and seed increase of self pollinated offspring and carefultesting of the genotype over several generations. This is an openpollinated method that works well with naturally self pollinatingspecies. This method can be used in combination with mass selection invariety development. Variations in pedigree and mass selection incombination are the most common methods for generating varieties in selfpollinated crops.

Hybrids.

A hybrid is an individual plant resulting from a cross between parentsof differing genotypes. Commercial hybrids are now used extensively inmany crops, including corn (maize), sorghum, sugarbeet, sunflower andbroccoli. Hybrids can be formed in a number of different ways, includingby crossing two parents directly (single cross hybrids), by crossing asingle cross hybrid with another parent (three-way or triple crosshybrids), or by crossing two different hybrids (four-way or double crosshybrids).

Strictly speaking, most individuals in an out breeding (i.e.,open-pollinated) population are hybrids, but the term is usuallyreserved for cases in which the parents are individuals whose genomesare sufficiently distinct for them to be recognized as different speciesor subspecies. Hybrids may be fertile or sterile depending onqualitative and/or quantitative differences in the genomes of the twoparents. Heterosis, or hybrid vigor, is usually associated withincreased heterozygosity that results in increased vigor of growth,survival, and fertility of hybrids as compared with the parental linesthat were used to form the hybrid. Maximum heterosis is usually achievedby crossing two genetically different, highly inbred lines.

The production of hybrids is a well-developed industry, involving theisolated production of both the parental lines and the hybrids whichresult from crossing those lines. For a detailed discussion of thehybrid production process, see, e.g., Wright, Commercial Hybrid SeedProduction 8:161-176, In Hybridization of Crop Plants.

RNA Interference (RNAi)

RNA interference (RNAi) is the process of sequence-specific,post-transcriptional gene silencing or transcriptional gene silencing inanimals and plants, initiated by double-stranded RNA (dsRNA) that ishomologous in sequence to the silenced gene. The preferred RNA effectormolecules useful in this invention must be sufficiently distinct insequence from any host polynucleotide sequences for which function isintended to be undisturbed after any of the methods of this inventionare performed. Computer algorithms may be used to define the essentiallack of homology between the RNA molecule polynucleotide sequence andhost, essential, normal sequences.

The term “dsRNA” or “dsRNA molecule” or “double-strand RNA effectormolecule” refers to an at least partially double-strand ribonucleic acidmolecule containing a region of at least about 19 or more nucleotidesthat are in a double-strand conformation. The double-stranded RNAeffector molecule may be a duplex double-stranded RNA foamed from twoseparate RNA strands or it may be a single RNA strand with regions ofself-complementarity capable of assuming an at least partiallydouble-stranded hairpin conformation (i.e., a hairpin dsRNA or stem-loopdsRNA). In various embodiments, the dsRNA consists entirely ofribonucleotides or consists of a mixture of ribonucleotides anddeoxynucleotides, such as RNA/DNA hybrids. The dsRNA may be a singlemolecule with regions of self-complementarity such that nucleotides inone segment of the molecule base pair with nucleotides in anothersegment of the molecule. In one aspect, the regions ofself-complementarity are linked by a region of at least about 3-4nucleotides, or about 5, 6, 7, 9 to 15 nucleotides or more, which lackscomplementarity to another part of the molecule and thus remainssingle-stranded (i.e., the “loop region”). Such a molecule will assume apartially double-stranded stem-loop structure, optionally, with shortsingle stranded 5′ and/or 3′ ends. In one aspect the regions ofself-complementarity of the hairpin dsRNA or the double-stranded regionof a duplex dsRNA will comprise an Effector Sequence and an EffectorComplement (e.g., linked by a single-stranded loop region in a hairpindsRNA). The Effector Sequence or Effector Strand is that strand of thedouble-stranded region or duplex which is incorporated in or associateswith RISC. In one aspect the double-stranded RNA effector molecule willcomprise an at least 19 contiguous nucleotide effector sequence,preferably 19 to 29, 19 to 27, or 19 to 21 nucleotides, which is areverse complement to the RNA of Camelina genes (e.g., FAD2 and FAE1genes), or an opposite strand replication intermediate. In oneembodiment, said double-stranded RNA effector molecules are provided byproviding to a Camelina plant, plant tissue, or plant cell an expressionconstruct comprising one or more double-stranded RNA effector molecules.In one embodiment, the expression construct comprises a double-strandRNA derived from any one of SEQ ID NOs 1-6 and SEQ ID NOs 45-63. Inother embodiments, the expression construct comprises a double-strandRNA derived from more than one sequences of SEQ ID NOs 1-6 and SEQ IDNOs 45-63. In further embodiments, the expression construct comprises adouble-strand RNA derived from more than one sequences of SEQ ID NOs 1-6and SEQ ID NOs 45-63, and one or more other genes involved in plantfatty acid synthesis. One skilled in the art will be able to designsuitable double-strand RNA effector molecule based on the nucleotidesequences of Camelina FAD2 and FAE1 in the present invention and otherCamelina fatty acid synthesis genes known in the art.

In some embodiments, the dsRNA effector molecule of the invention is a“hairpin dsRNA”, a “dsRNA hairpin”, “short-hairpin RNA” or “shRNA”,i.e., an RNA molecule of less than approximately 400 to 500 nucleotides(nt), or less than 100 to 200 nt, in which at least one stretch of atleast 15 to 100 nucleotides (e.g., 17 to 50 nt, 19 to 29 nt) is basedpaired with a complementary sequence located on the same RNA molecule(single RNA strand), and where said sequence and complementary sequenceare separated by an unpaired region of at least about 4 to 7 nucleotides(or about 9 to about 15 nt, about 15 to about 100 nt, about 100 to about1000 nt) which forms a single-stranded loop above the stem structurecreated by the two regions of base complementarity. The shRNA moleculescomprise at least one stem-loop structure comprising a double-strandedstem region of about 17 to about 500 bp; about 17 to about 50 bp; about40 to about 100 bp; about 18 to about 40 bp; or from about 19 to about29 bp; homologous and complementary to a target sequence to beinhibited; and an unpaired loop region of at least about 4 to 7nucleotides, or about 9 to about 15 nucleotides, about 15 to about 100nt, about 250-500 bp, about 100 to about 1000 nt, which forms asingle-stranded loop above the stem structure created by the two regionsof base complementarity. It will be recognized, however, that it is notstrictly necessary to include a “loop region” or “loop sequence” becausean RNA molecule comprising a sequence followed immediately by itsreverse complement will tend to assume a stem-loop conformation evenwhen not separated by an irrelevant “stuffer” sequence.

The expression construct of the present invention comprising DNAsequence which can be transcribed into one or more double-stranded RNAeffector molecules can be transformed into a Camelina plant, wherein thetransformed plant produces different fatty acid compositions than theuntransformed plant. The target sequence to be inhibited by the dsRNAeffector molecule include, but are not limited to, coding region, 5′ UTRregion, 3′ UTR region of fatty acids synthesis genes. In one embodiment,the target sequence is from one or more Camelina FAD2 and/or FAE1 genes.

The effects of RNAi can be both systemic and heritable in plants. Inplants, RNAi is thought to propagate by the transfer of siRNAs betweencells through plasmodesmata. The heritability comes from methylation ofpromoters targeted by RNAi; the new methylation pattern is copied ineach new generation of the cell. A broad general distinction betweenplants and animals lies in the targeting of endogenously producedmiRNAs; in plants, miRNAs are usually perfectly or nearly perfectlycomplementary to their target genes and induce direct mRNA cleavage byRISC, while animals' miRNAs tend to be more divergent in sequence andinduce translational repression. Detailed methods for RNAi in plants aredescribed in David Allis et al (Epigenetics, CSHL Press, 2007, ISBN0879697245, 9780879697242), Sohail et al (Gene silencing by RNAinterference: technology and application, CRC Press, 2005, ISBN0849321417, 9780849321412), Engelke et al. (RAN Interference, AcademicPress, 2005, ISBN 0121827976, 9780121827977), and Doran et al. (RNAInterference: Methods for Plants and Animals, CABI, 2009, ISBN1845934105, 9781845934101), which are all herein incorporated byreference in their entireties for all purposes.

The present invention is further illustrated by the following examplesthat should not be construed as limiting. The contents of allreferences, patents, and published patent applications cited throughoutthis application, as well as the Figures, are incorporated herein byreference in their entirety for all purposes.

EXAMPLE Example 1 Methods and Materials Southern Blot

Camelina sativa Cs32 and Cs11, and Arabidopsis thaliana ecotype Col-0(Table 2) seeds were germinated on Arabidopsis Growth Media (1×Murashige and Skoog (MS) mineral salts, 0.5 g/L MES, 0.8% PhytaBlend™all from Caisson Labs, North Logan, Utah; pH5.7) and allowed to grow for˜2 weeks under 16/8 hours day/night, 22/18° C., and ˜130 μE m⁻² s⁻¹light intensity. Genomic DNA was isolated according to the CTAB method(Saghai-Maroof, Soliman et al. 1984) and 10 μg was digested overnight(˜16 h) with EcoRI or a combination of EcoRI plus BamHI. DNAelectrophoresis and blotting were carried out using standard molecularbiology techniques (Tom Maniatis 1982). The probe was labelled withα-32P dCTP according to instructions of the DECAprime II kit (Ambion,Austin, Tex.). Hybridization was carried out overnight at 42° C. Theblot was washed (30 minutes each) at 42° C. in 2×SSC, 0.1% SDS, followedby 55° C. in 2×SSC, 0.1% SDS, and then 55° C. in 0.1×SSC, 1% SDS, andexposed to a phosphorimager screen. The blot was hybridized withdifferent probes after stripping the membrane in boiling 0.1% SDS for 20minutes each time.

Cloning of C. sativa FAD2 and FAE1 Genes and Upstream Regions.

FAD2 and FAE1 genes were amplified from C. sativa Cs32 DNA isolated asdescribed above, using Phusion polymerase (New England Biolabs, Ipswich,Mass.) and the primers listed in Table 3, according to themanufacturer's directions. The amplified fragments were cloned using theZero Blunt PCR Cloning kit (Invitrogen, Carlsbad, Calif.)

FAD2 and FAE1 Sequence Alignments

Translated amino acid FAD2 and FAE1 sequences were aligned with AlignX(Invitrogen), with a gap opening penalty of 15, a gap extension penaltyof 6.66, and a gap separation penalty range of 8. Alignments wereimported into Boxshade (EMBnet) to highlight the conserved residues.

RNA Isolation and cDNA Preparation

C. sativa Cs32 plants were grown under 24/18° C. day/night conditionswith a 16/8 hour photoperiod. Flowers were tagged and embryos harvestedat the time points indicated. RNA was then isolated using the urea LiClmethod described by Tai et al (Tai, Pelletier et al. 2004). cDNA wereprepared from 0.5 μg of DNAsed RNA that was reverse transcribed with theHigh Capacity cDNA RT kit (Applied Biosystems, Foster City, Calif.)using random primers according to the manufacturer's instructions.

Quantitative Real-Time PCR

Relative expression of FAD2 and FAE1 cDNA was measured by real-timequantitative PCR and calculated according to the comparative C_(T)method (2^(−ΔΔCT)). In brief, separate reactions were prepared induplicate or triplicate for each of the genes to be measured. Eachreaction contained 8 μl of the appropriate primers (200 nM each) andprobe (900 nM) for Cs ACTIN (reference gene) or Cs FAD2 or FAE1 (targetgene); 10 μl of Applied Biosystems 2× fast Taqman PCR mix; 2 μl of cDNA.The reactions were run on an Applied Biosystems 7900HT according to themanufacturer's fast PCR method. Real-time primers and probes are listedin Table 4.

Relative Expression Analysis

Three single nucleotide polymorphisms (SNPs) for each of FAD2 A, B, andC and FAE1 A, B, and C were identified. Each identified SNPdistinguishes one copy from the other two. An additional SNP, whichdistinguishes FAE1 A, B, and C copies from each other, was alsoidentified (Table 5). SNP frequencies were determined in cDNA isolatedas described above by the Sequenom MassARRAY™ allele-specific expressionanalysis method with no competitor, as described in Park et al (Park,Correll et al. 2004).

Genome Size Estimation

Camelina lines (Table 2) were grown in the greenhouse at temperaturesfluctuating between 16 and 26 C with 16 hour day length supplemented byhalogen lights. The nuclei were extracted from leaves according to Henryet al [74]. Nuclei were also extracted from approximately 50 seeds ofall species, except C. laxa and C. hispida, which are late flowering.The seeds were crushed with a pestle in 1.4 mL of the same extractionbuffer used for the leaves. The fluid was then drawn through four layersof cheesecloth and strained and processed as for the leaf nuclei. Nucleiof diploid and tetraploids of Arabidopsis thaliana accession Col-0 (1 Cgenome size 157 Mb, and 314 Mb, respectively [75]), and tetraploidArabidopsis arenosa accession Care-1 (1C genome size 480 Mb [Dilkes,unpublished results]) were used as standards for DNA content. Data wascollected on two different days and normalized separately to account fordaily fluctuations in flow cytometer performance. The 2C, 4C, and 8Cnuclear peaks were used in a regression analysis of measuredfluorescence intensity versus nuclear DNA content, producing equationsof genome size versus fluorescence that were used to estimate the 2Ccontent of Camelina nuclei.

Phylogenetic Inference

FAD2 and FAE1 were PCR amplified from several Camelina species and otherspecies from the tribe Camelineae (Table 2) using primers designed fromC. sativa FAD2 and FAE1 sequences (Table 3). Amplified fragments forFAD2 and FAE1 were cloned as described for C. sativa above, then alignedby translated amino acids sequences using MacClade 4.05 (Maddison 2004.)ModelTest 3.7 (Posada and Crandall 1998) in PAUP*4.0 b (Swofford 2001)was used to determine the model of sequence evolution favored by thedata for each gene. Subsequent maximum likelihood (ML) analyses wereperformed in PAUP*4.0 b using a heuristic search with tree bisectionreconnection (TBR) branch swapping. ML clade support using 100 bootstrapdata sets were assessed and this support is presented on the most likelytree recovered from the ML heuristic search.

Camelina Alkaline Transesterification for FAMES Composition and GasChromatography (GC/FID) Analysis of Camelina seeds

Approximately 50 mg of seeds were ground up in liquid nitrogen withmortar and pestle. 5 mL of 0.2M KOH in methanol was added to each vialcontaining the ground seeds. Samples were capped, heated at 37 C. for 1hr and vortexed every 10 minutes. Reaction was stopped with addition of1 mL 1M acetic acid and 2 mL heptanes. Samples were vortexed, and thencentrifuged for 10 min at room temp at 2990 rpm and the upper organicphase was collected. Before GC analysis, samples were diluted 1/10 inheptanes.

The supernatant was transferred to a GC vial, in which 1 μL was used forGC analysis. Analysis was carried out on GC/FID 7890A series with aSP_(—)2330 column. Injector and detector temperature were 250° C. and300° C. respectively; oven temperature was held at 50° C. for 2 min,then programmed to 180° C. at a heating rate of 10° C./min, thenprogrammed to hold for 5 min followed by an increase of 5° C./min to240° C. Total run time was 32.5 min. Flow rates for hydrogen and air tothe FID were 30 and 450 mL/min respectively. Helium as the carrier gasflowed at a rate of 1.69 mL/min and nitrogen as the make-up gas at 30mL/min.

Example 2 Southern Blot Hybridizations Show Multiple Copies of Genes inCamelina Sativa

As a first step to characterize genes involved in fatty acidbiosynthesis, the inventors determined the copy number of FAD2 and FAE1by Southern blot analysis. Since C. sativa is closely related toArabidopsis thaliana (Al-Shehbaz, Beilstein et al. 2006; Beilstein,Al-Shehbaz et al. 2006; Beilstein, Al-Shehbaz et al. 2008), theinventors designed primers based on Arabidopsis that amplified conservedregions of FAD2 and FAE1. Using these primers, the inventors PCRamplified products of 225 base pairs (bp) (FAD2) and 403 by (FAE1) fromArabidopsis and from C. sativa. The C. sativa products were cloned,sequenced, and compared with Arabidopsis FAD2 and FAE1 sequences (TAIR2009) to confirm their identities. The inventors used the C. sativafragments as probes in Southern blot experiments (FIG. 1). Results ofthe Southern blots revealed three bands in C. sativa for both FAD2 (FIG.1A) and FAE1 (FIG. 1B), whereas hybridization revealed only a singleband in Arabidopsis for both genes (FIGS. 1A & B). These results suggestthat FAD2 and FAE1 occur in at least three copies in C. sativa, whilethey are single copy in Arabidopsis (TAIR 2009). Fatty acid genes can bemulti-copy in many species, including soybean (Schlueter, Lin et al.2007), Brassica napes (Scheffler, Sharpe et al. 1997), olive (Oleaeuropaea) (Hernandez, Mancha et al. 2005), maize (Mikkilineni andRocheford 2003), and sunflower (Martinez-Rivas, Sperling et al. 2001).Therefore, the inventors designed a probe for Southern blothybridization of the gene LEAFY (LFY), which is known to be single copyin a wide variety of species from several plant families (Frohlich andEstabrook 2000). Three bands were observed following hybridization usingthe LFY probe, suggesting LFY also exists as three copies in C. sativa(FIG. 1C).

Example 3 Copies of C. Sativa FAD2 and FAE1 are Highly Similar to EachOther and to their Putative Orthologs from Arabidopsis

The inventors cloned and sequenced the full length genomic and cDNAsequences of C. sativa FAD2 and FAE1 (SEQ ID NOs: 1 to 6). Using primersdesigned from Arabidopsis FAD2 and Crambe abyssinica FAE1, the inventorsPCR amplified a band of approximately 1.2 kb for FAD2 and 1.5 kb forFAE1 from C. sativa. For each gene, the inventors sequenced more than 60clones. Three different versions of both FAD2 and FAE1 were recoveredand designated A, B, and C. It should be noted that the A, B, and Ccopies were named independently for FAD2 and FAE1, and thus are notassociated with a particular genome.

The three copies of C. sativa FAD2 are 1155 by long, lack introns in thecoding regions, are 97% identical at the nucleotide level, and encodeproteins that are 99% identical in sequence (Table 1). One of the FAD2copies contains a BamHI site, and thus this copy likely produced the˜1.3 kb fragment in the Southern blot hybridization of FAD2 (FIG. 1A;BamHI+ EcoRI digest). The C. sativa nucleotide sequences of FAD2 aregreater than 93% identical to Arabidopsis FAD2, and the putative encodedproteins from the two species share greater than 96% identity (Table 1).

An approximately 1.4 kb intron found within the 5′ untranslated regionwas also recovered from all three copies of C. sativa FAD2. A similarlysized intron is present in Arabidopsis (TAIR 2009) and in Sesamumindicum (sesame) where it has been shown to be involved in regulatingFAD2 expression (Kim et al. 2006).

All three copies of FAE1 in C. sativa are 1518 by long and lack introns.When the nucleotide sequences and the putative encoded proteins of thethree copies are compared they are more than 96% identical (Table 1). Incomparison to Arabidopsis, the nucleotide sequences are more than 90%identical, while the encoded proteins are more than 91% identical (Table1). Thus, the three copies of C. sativa FAD2 and the three copies ofFAE1 are highly similar to each other and to their putative orthologsfrom Arabidopsis.

Example 4 Alignments of FAD2 and FAE1 Protein Sequences from SeveralSpecies Reveals Conserved and Non-Conserved Domains

The inventors aligned translated amino acid sequences from the threecopies of C. sativa FAD2 with the FAD2 protein sequences fromArabidopsis; Brassica rapa, an agronomically important member of theBrassicaceae family; Glycine max, an agronomically important dicot; andZea mays, an agronomically important monocot (FIG. 2A). All three copiesof C. sativa FAD2 have the three conserved HIS boxes found in allmembrane-bound desaturases (Tocher D R 1998) as well as the ERlocalization signal described by McCartney et al (Belo, Zheng et al.2008)(McCartney, Dyer et al. 2004). Furthermore, the conserved aminoacids identified in an alignment of the FAD2 sequences from 34 differentspecies [49] are also present in C. sativa with the exception of apositively-charged histidine at position number 44, which is substitutedby a polar, uncharged glutamine in C. sativa. When the inventorsamplified the FAD2 gene from several species in the tribe Camelineae(Table 2) and aligned the translated amino acid sequences, the inventorsfound that the FAD2 proteins from Capsella rubella, Camelina microcarpa,Camelina laxa, and one copy from Camelina rumelica contain a glutamineat amino acid position 44, while the FAD2 proteins from Arabidopsislyrata, Camelina hispida, and a second copy from Camelina rumelicacontained a histidine (data not shown).

TABLE 1 Nucleotide and Amino Acid identity of Camelina sativa andArabidopsis thaliana FAD2 and FAE1 genes. Gene % Nucleotide Identity* %Amino Acid Identity AtFAD2 CsFAD2A CsFAD2B CsFAD2C AtFAD2 CsFAD2ACsFAD2B CsFAD2C FAD2 AtFAD2 100 93.6 93.8 93.4 100 96.9 96.6 96.4CsFAD2A 100 97.3 98.3 100 99.0 99.5 CsFAD2B 100 97.7 100 99.5 CsFAD2C100 100 AtFAE1 CsFAE1A CsFAE1B CsFAE1C AtFAE1 CsFAE1A CsFAE1B CsFAE1CFAE1 AtFAE1 100 90.7 91.2 91.0 100 91.9 91.7 91.7 CsFAE1A 100 97.8 96.8100 97.6 96.4 CsFAE1B 100 97.2 100 96.8 CsFAE1C 100 100 *Nucleotideidentity is in coding region only.

TABLE 2 Plant species and sources Species Source Catalogue numberCamelina sativa Cs32 USDA PI 311732 Camelina sativa Cs11 Ames 26668Arabidopsis thaliana, ABRC CS28166 ecotype Col-0 Arabidopsis lyrata ABRCCS22696 Camelina laxa USDA PI 650132 Camelina microcarpa wildcollection; number “01-22” Harvard Herbarium Camelina microcarpa USDA PI633188 Capsella bursa pastoris Wild collection; number “08-188” HarvardHerbarium collection Capsella rubella ABRC CS22561 Camelina hispida varAmes 21324 grandiflora Camelina alyssum Ames 26658 Camelina rumelicaAmes 21327

TABLE 3 Primers used for amplification of genomic regions of C. sativaPrimer Name Primer sequence (5′-3′) Southern FAD2_631FTCAACAACCCTCTTGGACGCATCA analysis of (SEQ ID NO: 13) FAD2 FAD2_832RCTTGTGCAGCAGCGTAACGGTAAA (SEQ ID NO: 14) Southern AtFAE1 probe FAGACGGTCCAAGTACAAGCTAGTTC analysis of (SEQ ID NO: 15) FAE1AtFAE1 probe R CCAAATCTATGTAACGTTGATCT (SEQ ID NO: 16) SouthernAtLFY probe F GATGCGGCGGGGAATAACGGCGGAG analysis of (SEQ ID NO: 17) LFYAtLFY probe R CCTGAAGAAGGAACTCACGGCATT (SEQ ID NO: 18) Cloning ofAtFAD2_start AACATGGGTGCAGGTGGAAGAATG FAD2 coding (SEQ ID NO: 19) regionAtFAD2_stop2 TCATAACTTATTGTTGTACCAGTAC (SEQ ID NO: 20) Cloning ofCaFAE1 start ATGACGTCCATTAACGTAAAGCTC FAE1 coding (SEQ ID NO: 21) regionCaFAE1 stop TTAGGACCGACCGTTTTGGGC (SEQ ID NO: 22) KCS17-FAE1 AtKCS FGGGTGGCTCTTCGCAATGTCGAGCCC intergenic (SEQ ID NO: 23) region “A” andCsFAE1 5′ RACE GAGGCTTTTCCGGCAAGTAACGCCG “C” (initial (SEQ ID NO: 24)clones) KCS17-FAE1 AtKCS cons F GGTATGAATTGGCTTACACGGAAG intergenic(SEQ ID NO: 25) region “A” CsKCSA_F TATGAATTGGCTTACACGGAAGCC(SEQ ID NO: 26) CsFAE1A_R2 TATATTGCCAATATAAGTATTAAAGGTCC (SEQ ID NO: 27)KCS17-FAE AtKCS cons F GGTATGAATTGGCTTACACGGAAG intergenic(SEQ ID NO: 28) region “B” CsFAE1B_R TATATTGCCAATATAAGTATTAAAGGTCC(SEQ ID NO: 29) KCS17-FAE AtKCS cons F GGTATGAATTGGCTTACACGGAAGintergenic (SEQ ID NO: 30) region “C” CsFAE1C_R GGTAGAGATCGTTTGTGGTAAGCG(SEQ ID NO: 31) Camelinae CsFAD2 start ATGGGTGCAGGTGGAAGAATGC FAD2(SEQ ID NO: 32) CsFAD2 stop TCATAACTTATTGTTGTACCAGTACACACC(SEQ ID NO: 33) Camelinae CsFAE1 start ATGACGTCCGTTAACGCAAAGCTC FAE1(SEQ ID NO: 34) CsFAE1 stop TTAGGACCGACCGTTTTTGACATG (SEQ ID NO: 35)

TABLE 4 Primers used for qPCR analyses Primer or Probe NameSequence (5′-3′) qPCR of CsACT For ACA ATT TCC CGC TCT GCT GTT GTGCsACTIN (SEQ ID NO: 36) CsACT Rev AGG GTT TCT CTC TTC CAC ATG CCA(SEQ ID NO: 37) CsACT probe FAM - TGT TTC AAA CGC TCT ATC CCTCGC TC - IABLFQ (SEQ ID NO: 38) qPCR of CsFAD2 A For1CTG CGA GAA ACC ACC GTT CAC CC CsFAD2 (SEQ ID NO: 39) CsFAD2 all RevCAC GAG TAG TCA ACG AGG TAA ACC GG (SEQ ID NO: 40) CsFAD2 all FAM - CCA CTT CTA TTC CCA TCT CCA probe ACA CAA CC - IABLFQ(SEQ ID NO: 41) qPCR of CsFAE1 all For AAC CTT TGC TTG TTT CCG TTA ACGCsFAE1 GC (SEQ ID NO: 42) CsFAE1 all Rev CAC GAG TAG TCA ACG AGG TAAACC GG (SEQ ID NO: 43) CsFAE1 all  FAM - CCA CTT CTA TTC CCA TCT CCAprobe ACA CAA CC - IABLFQ (SEQ ID NO: 44)

TABLE 5 SNPs distinguishing each copy of FAD2 and FAE1 Nucleotideposition from beginning of SNP_ID coding region FAD2_A4 51 FAD2_A2 453FAD2_A6 549 FAD2_B4 288 FAD2_B5 687 FAD2_B8 1109 FAD2_C1 78 FAD2_C5 615FAD2_C3 966 FAE1_A4 624 FAE1_A3 1368 FAE1_A7 1475 FAE1_B4 414 FAE1_B5783 FAE1_B8 1438 FAE1_C1 336 FAE1_C2 721 FAE1_C7 1419 FAE1_ABC1 104

The inventors aligned the translated amino acid sequences from the threecopies of C. sativa FAE1 with the seed-specific FAE1 proteins from A.thaliana, Crambe abyssinica, a high and low erucic acid Brassica rapa,Limnanthes alba, and Tropaeolum majus (FIG. 2B). L. alba and T. majusare both in the order Brassicales and their seeds accumulate high levelsof very long chain fatty acids (Cahoon, Marillia et al. 2000;Mietkiewska, Giblin et al. 2004). Four conserved histidine residues andsix conserved cysteine residues, including the active site at cysteine223, as well as an asparagine residue at 424 required for FAE1 activitywere previously identified by Ghanevati and Jaworski (Ghanevati andJaworski 2001; Ghanevati and Jaworski 2002). All conserved residues werefound to be present in all three copies of C. saliva FAE1. Moredifferences were apparent between the three C. saliva FAE1 sequences andthe other FAE1 sequences than observed in the FAD2 comparison (FIGS. 2Aand B), an observation consistent with the level of amino acid identityseen between Arabidopsis and C. saliva FAD2 versus FAE1 (Table 1).

Example 5 All Three Copies of FAD2 and FAE1 are Expressed in DevelopingSeeds of C. Sativa

The conservation of amino acids as well as the presence of the 5′regulatory intron in FAD2 suggests that all three copies of FAD2 and ofFAE1 could be functional. To determine whether these genes are alsoexpressed, the inventors first evaluated total FAD2 and FAE1 geneexpression in developing seeds and in seedling tissue using quantitativereal time PCR (qPCR) with primer/probe combinations designed to detectall three copies of each gene. FAD2 expression in seedling tissue ispresent but minimal (0.4% of that seen in seeds at 20 days post-anthesis(DPA)), while FAE1 expression could not be detected in seedlings (FIGS.3A and B). In developing seeds, both FAD2 and FAE1 expression peaks at20 DPA and is reduced by 30 DPA (FIGS. 3A and B). In Arabidopsis, FAD2peaks earlier and decreases sooner than FAE1 (Ruuska, Girke et al.2002).

The inventors wondered whether the expression of each of the FAD2 andFAE1 copies present in C. sativa are equally or differentially expressedin the seed. Duplicated genes are frequently silenced either throughoutthe plant or in a tissue-specific manner (Comai, Tyagi et al. 2000;Kashkush, Feldman et al. 2002; He, Friebe et al. 2003; Adams, Percifieldet al. 2004); hence the inventors hypothesized that one or more of thecopies of each gene could be significantly down-regulated. The inventorsused the Sequenom MassARRAY™ method for determining allele-specificexpression of a gene (Park, Correll et al. 2004) to evaluate therelative expression of each of the copies of FAD2 and FAE1. Theinventors identified at least three single nucleotide polymorphisms(SNPs) specific to each of the FAD2 A, B, and C and the FAE1 A, B, and Ccopies and then calculated the frequency of each SNP in seed cDNA.Controls consisting of the cloned FAE1 A, B, and C copies combined toknown frequencies showed that the method is greater than 80% accurate(Table 6). No evidence of silencing of any particular copy of eitherFAE1 or FAD2 was discovered. The inventors did observe differentialexpression, especially of FAE1 A, which accounts for approx 40-50% ofFAE1 expression in seeds at 20-30 DPA (FIGS. 3C and D).

Six cloned DNA positive controls were also included in the analysis andthe relative amount of “B” version in each measured with the FAE1_B5 SNPand all 3 versions with the FAE1_ABC SNP:

TABLE 6 Expression level of FAE1 genes relative to FAE1 B FAE1_B5 FAE1ABC relative relative relative relative B A B C C1 100% Version A 0.00 10 0 C2 100% Version B 1.00 0 1 0 C3 100% Version C 0.00 0 0 1 C4 60% A,20% B, 20% C 0.20 0.59 0.20 0.22 C5 20% A, 60% B, 20% C 0.54 0.29 0.480.23 C6 20% A, 20% B, 60% C 0.20 0.24 0.28 0.48

As the results indicate, all three FAE1 genes are expressed in the seed.A dosage effect may still be expected, however, since FAE1 B appears toaccount for only approximately 25-30% of FAE1 expression in the seeds. Amutation in FAE1 A would be expected to have a greater effect on fattyacids composition in the seeds since it accounts for ˜41-48% of FAE1expression.

Example 6 Characterization of Sequences Upstream of C. Sativa FAE1 andDownstream of C. Sativa FAD2 Suggests Colinearity with A. Thaliana

To investigate whether the different copies of C. sativa FAD2 and FAE1are the result of allelic variation or are in fact independent loci, theinventors obtained sequence from the region upstream of FAE1 anddownstream of FAD2. Assuming colinearity between C. sativa andArabidopsis for the region around FAE1, the inventors PCR amplified theregion 5′ to FAE1 using a forward primer for the upstream gene KCS17with reverse primers for C. sativa FAE1. The resulting sequencesobtained for the putative C. saliva KCS17 were highly similar to thelast 189 bp of Arabidopsis KCS17, suggesting that the inventors had infact amplified the orthologous C. sativa region upstream of FAE1,confirming colinearity between the two species. The inventors then useda dot plot (see details for Nucleic Acid Dot Plots in Maizel et al.,1981; Pustell et al. 1982; Quigley et al., 1984) to compare the three C.sativa upstream sequences to each other and to Arabidopsis withparameters set for perfect match on a sliding window of 9 bases. Thecoordinates from the dot plot were used to define blocks of homologybetween Arabidopsis and the three C. sativa copies (FIG. 4). The resultsshow a variable intergenic region containing conserved blocks common totwo or more genomes.

Co-linearity with Arabidopsis was also found for a region downstream ofFAD2 containing the ACTIN11 (ACT11) gene for two out of the three C.sativa copies (data not shown). For the third copy, the regiondownstream of FAD2 A could have been missed if the length of theamplified product was too large. Alternatively, the region downstream ofFAD2 A might not exhibit colinearity with Arabidopsis.

Example 7 The Genomes of C. Sativa, C. Alyssum, and C. Microcarpa areLarger than the Genomes of Other Camelina Species

The inventors calculated DNA content in several accessions of C. sativaand related species from flow cytometry analyses using propidiumiodide-stained nuclei. The inventors used Arabidopsis accession Col-0(2×) and its tetraploid (4×) derivative as genome size standards. C.sativa, C. alyssum, and C. microcarpa diploid (2C) genomes had a haploidcontent between 650 and 800 Mb (FIG. 5). C. sativa accessions uniformlydisplayed a genome size close to 750 Mb. North American isolates of C.sativa, C. alyssum, and C. microcarpa have reported chromosome counts ofn=20 (Francis and Warwick 2009). The genomes of C. rumelica (600 Mb), C.hispida (300 Mb) and C. laxa (210 Mb) are smaller than those of C.sativa, C. alyssum, and C. microcarpa. Chromosome counts of both n=6(Baksay 1957; Brooks 1985) and n=12 (Maassoumi 1980) have been recordedfor C. rumelica, while only a single count of n=7 exists for C. hispida(Maassoumi 1980). To our knowledge, no published counts exist for C.laxa.

Example 8 Phylogenetic Analysis of FAD2 and FAE1 Indicate that C. Sativaand C. Microcarpa are Closely Related

To understand the duplication history of the multiple FAD2 and FAE1copies recovered from C. sativa, the inventors amplified the FAD2 andFAE1 genes from several species in the tribe Camelineae (Table 2), andinferred phylogeny for each gene. The sampling of taxa chosen allowedthe inventors to test whether FAD2 and FAE1 duplication events occurredafter Camelina diverged from its closest relatives or within the genus.Results from the evaluation of 55 different models of sequence evolutionusing Modeltest 3.7 (Posada and Crandall 1998) indicated that the FAD2sequence data are best described by the TVM+I+Γ model, while the FAE1data are best described by the HKY+I+Γ model. Likelihood phylogeneticanalyses in PAUP*4.0 (Swofford 2001) produced a single FAD2 tree (−LnL3665.277; FIG. 6A), and a single FAE1 tree (−LnL 5051.552; FIG. 6B).

Phylogenies inferred from FAD2 and FAE1 data indicate a history ofduplication for both markers. Both C. microcarpa and C. sativa havethree distinct copies of FAD2 and FAE1. Moreover, for FAD2, the A and Ccopies from these two species are monophyletic with strong (100%)bootstrap support (bs); for FAE1 the A and B copies from these speciesare strongly monophyletic (100% bs). In contrast, neither the FAD2 Bcopies of C. sativa and C. microcarpa, nor the FAE1 C copies of thesespecies form a monophyletic group with each other. Instead, resultsindicate that C. rumelica has two distinct copies of FAD2 and that oneof these copies (FAD2-2) is strongly monophyletic with C. microcarpaFAD2 B. The inventors recovered only a single FAD2 copy for C. laxa andC. hispida. In contrast, at least two distinct copies of FAE1 wererecovered from all sampled Camelina species. The FAE1-1 copy of C. laxa,C. hispida, and C. rumelica form a monophyletic group (91% bs), with theformer two species sister to one another with strong support (100% bs).Similar to the results from FAD2, C. rumelica FAE1-2 is sister to one ofthe C. microcarpa copies (FAE1 C; 99% bs). Neither the C. sativa FAD2 Bcopy, nor the C. sativa FAE1 C copy, shows a well supported sisterrelationship to other FAD2 or FAE1 sequences. However, in the FAE1 tree,C. sativa FAE1 C is very weakly supported as sister to C. hispida FAE1-2(53%). Finally, all recovered FAD2 and FAE1 copies from species of thegenus Camelina are monophyletic and sister to other sampled members ofthe tribe Camelineae, consistent with phylogenies based on other markers(Beilstein, Al-Shehbaz et al. 2006; Beilstein, Al-Shehbaz et al. 2008).

Example 9 Camelina Breeding Program

Since Camelina has not been intensively bred and the germplasm issomewhat limited genetically, the inventors established three strategiesfor long term development of Camelina germplasm. These three,non-mutually exclusive strategies for Camelina germplasm enhancementinclude: transgenic approach, classical and molecular breeding, andmutation breeding. The long term goals are to achieve increased yield,increased seed oil content and improved fatty acid composition (e.g.,higher percentage oleic acid (18:1), which is an optimal fatty acid forbiodiesel and/or lower percentage of very long chain fatty acids (VLCFA,such as 20:1, 20:2, 22:1, etc)).

In the transgenic approach, REV and KRP yield technology (US 2008/263727and US 2007/056058, incorporated by reference in their entireties) canbe introduced into Camelina to obtain events with increased seed yieldor seed size, agronomic properties beneficial to obtaining Camelinagermplasm with increased oil yield per unit land for biofuel purposes.Efficient transformation of Camelina has been established before (WO2009/117555, incorporated by reference in its entirety).

In the classical and molecular breeding approach, broad fieldevaluations of more than 100 accessions of Camelina in Northern UnitedStates and Canada was initiated across multiple field locations and overmultiple years. Different accessions were evaluated for seed yield, oilyield, fatty acid composition, and agronomic performance under differentenvironmental conditions. Superior lines with higher yield identified inthe evaluations are used in the breeding program. In addition, molecularbreeding studies are also in progress. Preliminary results show thatexisting Camelina cultivars are closely related, as indicated by AFLPanalysis in which 379 markers were scored. Jaccard analysis suggestedthere is more than 90% genetic similarity across existing cultivars.Therefore, there is much room for improvement of Camelina germplasm,which will be realized by classical and molecular breeding programs. Inthe mutation breeding approach, an EMS mutagenized population wascreated in a selected Camelina cultivar, and Targeting Induced LocalLesions In Genomes (TILLING®) method was used to find mutations in knowngene sequences. Especially, mutations with altered fatty acidcompositions and improved yield as expressed in amount of oil producedper acre are of the most interest. M2 plants/M3 seed were harvested, andgene sequences for select targets were isolated and characterized.Preferred fatty acids include 16:1 and 18:1 monounsaturated fatty acids,since they have the best combination of proper cetane number, cloudpoint, oxidative stability, and less NOx emissions, as compared tosaturated fatty acids (e.g., 12:0, 14:0, 16:0, 18:0, 20:0, and 22:0), orpoly unsaturated fatty acids (e.g., 18:2, 18:3).

Example 10 TILLING® Method to Isolate Camelina Mutants in FAD2 and FAE1Genes

As described above, the goal is to improve Camelina sativa fatty acidcomposition for biodiesel. For example, since oleic acid (18:1) isoptimal for fatty acid biodiesel, one specific goal is to increase 18:1and decrease polyunsaturated fatty acids and long chain fatty acids. Oneway is to lower the activity of FAD2 and of FAE1, as indicated by thefatty acids synthesis pathway shown in FIG. 7.

An EMS mutant library has been created in Camelina sativa line CS32.This library has a population of about 8000 mutants and was used toscreen for mutants of FAD2 genes (FIG. 8). Initial TILLING® usingprimers designed to the three FAD2 genes yielded mutants in all threeFAD2 genes. Later, TILLING® using primers designed to the three FAE1genes also yielded mutants in all three FAE1 genes. Lu et al (Camelinasativa: A Potential Oilseed Crop for Biofuels and Genetically EngineeredProducts, Information Systems for Biotechnology New Report, January2008) describes a preliminary mutant screen where a random screen wascarried out for fatty acid composition Camelina mutant using gaschromatography (GC). The TILLING® method of the present invention issuperior to this because it is not necessary to GC screen thousands ofmutants; rather, mutants in known fatty acid genes are identified(Hutcheon et al., TILLING® for Altered Fatty Acid Profiles in Camelinasativa, July 2009, American Society of Plant Biologists Annual Meeting,which is herein incorporated by reference in its entirety for allpurposes). Also the identification of Camelina sequences allows for thedesign of gene-specific TILLING® primers which can make it much easierto get mutations in all three versions of any given gene, FAD2 or FAE1.

A non-limiting exemplary protocol of TILLING® is described below:

-   -   1. Seeds are mutagenized to induce point mutations throughout        the genome.    -   2. A founder population is grown from mutagenized seeds.    -   3. Founder population is self-fertilized to produce a crossed        population.    -   4. DNA samples from the crossed population are collected in        96-well plates and seeds from the crossed population are stored.    -   5. Up to eight 96-well plates are pooled into one and the        samples (768) subjected to PCR with two gene-specific primers        labelled with different IRDye® infrared dyes.    -   6. Resulting amplicons are heated and cooled, resulting in        heteroduplexes between wild type and mutant samples.    -   7. CEL I nuclease is used to cleave at base mismatches.    -   8. Samples are denatured and electrophoresed on a LI-COR® DNA        Analysis System.    -   9. In lanes that have a mutation in the pool, a band will be        visible below the wild type band on the IRDye® 700 infrared dye        image. A counterpart band will be visible in the same lane on        the IRDye® 800 infrared dye image. This band is the cleavage        product labeled with IRDye® 800 infrared dye from the        complementary DNA strand. The sum of the length of the two        counterpart bands is equal to the size of the amplicon, which        makes it easy to distinguish mutations from amplification        artefacts. An exemplary LI-COR gel identifying mutants in        Camelina FAD2 genes is shown in FIG. 9.    -   10. After detection of a mutation in a pool (lane), the        individual DNA samples in the pool are screened again to find        out which of the eight pooled samples from the crossed        population has the mutation.

More information on TILLING® is described by Colbert et al. (2001. HighThroughput Screening for Induced Point Mutations. Plant Physiology 126:480-484.); McCallum et al. (2000. Target Induced Local Lesions InGenomes (TILLING) for Plant Functional Genomics. Plant Physiology123:439-442); Henikoff et al. (Single-Nucleotide Mutations for PlantFunctional Genomics. Annual Review of Plant Biology. 54:15.1-15.27.);and Till et al. (2003. Large-Scale Discovery of Induced Point MutationsWith High-Throughput TILLING. Genome Research 13:524-530).

A pilot study determined that the mutation density of the inventors'mutant Camelina population was 1/25 kb. TILLING® of an initial 768 M2individuals for FAD2 has identified 60 mutants, 60% of which arenon-silent mutations. Of the non-silent mutations, about 30% arepredicted to be severe missense or truncation mutations. Mutations wereidentified in all 3 copies of Camelina FAD2. The inventors' previousfinding that Camelina sativa may be polyploid is further supported bythe high density of lesions this plant is willing to tolerate in itsgenome. The mutant M3 plants were grown and a preliminary analysis oftheir fatty acid profiles by GC was performed.

Example 11 Mutations of Camelina FAD2 and FAE1 Genes Identified inTILLING®

Initial screening of the TILLING® population for FAD2 mutants resultedin plants with silent, STOP (nonsense) and/or severe missense mutationsin FAD2 A, B, and C; and FAE1 A, B and C genes.

Positions and effects of mutations in FAD2 A, B, and C genes and FAE1 A,B and C genes are displayed in Tables 7 to 12 below (*indicates themutation results in a stop codon,=indicates silent mutation).

TABLE 7 Summary of Camelina FAD2 A mutants Nucleotide Change EffectPrimer set Plant ID Mutation Score G1516A G35R FAD2A 2480 severemissense C1645T L78F FAD2A 2487 severe missense C1746T H111= FAD2A 2782silent C1813T P134S FAD2A 2085 severe missense G1844A R144H FAD2A 2764severe missense C1977T V188= FAD2A 2484 silent G2015A G201D FAD2A 2993severe missense C2099T S229F FAD2A 2579 severe missense G2155A G248RFAD2A 2200 severe missense G1495A, E28K, E287K FAD2A 2983 missense,severe G2272A missense G2138A R242H FAD2A 2986 missense

TABLE 8 Summary of Camelina FAD2 B mutants Nucleotide Change EffectPrimer set Plant ID Mutation Score C207T S53F FAD2B 2474 or 2199 severemissense C213T S55F FAD2B 3142 Severe Missense G785A A246T FAD2B 3363Missense C476T R143C FAD2B 3314 Severe Missense C176T P43S FAD2B 3325Severe Missense G462A W138* FAD2B 3489 Nonsense G498A G150E FAD2B 3702Severe Missense G779A A244T FAD2B 3732 Missense G737A D230N FAD2B 3814Missense C812T L255F FAD2B 4245 Missense C882T P278L FAD2B 4408 MissenseG410A D121N FAD2B 4875 Missense G675A C209Y FAD2B 4916 Missense C459TS137F FAD2B 5155 Severe Missense C528T P160L FAD2B 5746 Severe MissenseC987T T313M FAD2B 6023 Severe Missense C284T P79S FAD2B 6107 SevereMissense G416A V123I FAD2B 6122 Severe Missense G650A G201S FAD2B 6105Severe Missense C656T P203S FAD2B 6277 Missense C203T R52C FAD2B 6493Severe Missense G582A G178E FAD2B 6486 Severe Missense G372A C108Y FAD2B6479 Severe Missense G322A W91* FAD2B 6490 Nonsense G374A G109S FAD2B6752 Severe Missense G926A G293R FAD2B 6778 Severe Missense C490T S147=FAD2B 3207 silent C940T T297= FAD2B 3423 silent G148A T33= FAD2B 3521silent

TABLE 9 Summary of Camelina FAD2 C mutants Nucleotide Change EffectPrimer set plant ID Mutation Score G1429A E28K FAD2C 6431 MissenseC1501T R52C FAD2C 3168 Severe Missense C1542T S65= FAD2C 5756 silentC1576T L77F FAD2C 5550 Missense C1582T P79S FAD2C 5655 Severe MissenseG1607A W87* FAD2C 4506 Nonsense C1609T P88S FAD2C 3210 Severe MissenseG1619A W91* FAD2C 3284 Nonsense G1672A G109S FAD2C 3690 Severe MissenseG1717A G124S FAD2C 5644 Severe Missense C1720T L125F FAD2C 4933 MissenseC1741T L132F FAD2C 4995 Missense G1795A G150R FAD2C 3147 Severe MissenseG1796A G150E FAD2C 4608 Severe Missense C1799T S151F FAD2C 3275 SevereMissense G1808A R154K FAD2C 3490 Missense G1810A D155N FAD2C 2578, 2586Severe Missense C1857T G170= FAD2C 4716 silent C1873T P176S FAD2C 3267Severe Missense G1880A G178E FAD2C 5903 Severe Missense G1883A R179HFAD2C 4846 Severe Missense G1890A M181I FAD2C 4400 Missense G1915A G190RFAD2C 5524 Severe Missense G1948A G201S FAD2C 6120 Severe MissenseG1963A G206R FAD2C 4556 Missense C2029T L228F FAD2C 4802 Missense G2072AR242H FAD2C 5122 Missense G2080A A245T FAD2C 3152 Missense C2081T A245VFAD2C 5318 Missense C2084T A246V FAD2C 4884 Missense C2096T A250V FAD2C3318 Missense C2110T L255F FAD2C 5734 Missense C2112T L255= FAD2C 4677silent G2117A G257E FAD2C 5491 Severe Missense G2117A G257E FAD2C 6470Severe Missense G2140A A265T FAD2C 3924 Missense G2149A V268I FAD2C 6068Severe Missense C2188T P281S FAD2C 4864 Severe Missense C2204T S286FFAD2C 5183 Severe Missense G2255A G303E FAD2C 4467 Severe MissenseG2268A K307= FAD2C 6509 silent C2285T T313M FAD2C 5426 Severe MissenseC2293T H316Y FAD2C 2785, 2487, Severe Missense 2488, or 2786 C2315TS323L FAD2C 6060 Severe Missense G2422A E359K FAD2C 4997 Severe MissenseG2443A V366I FAD2C 6579 Missense C1595T S83F FAD2C 4138 Severe MissenseC2383T Q346* FAD2C 6077 Nonsense

TABLE 10 Summary of Camelina FAE1 A mutants Nucleotide Change EffectPrimer set Plant ID Mutation Score G621A V55I FAE1-A 4696 Missense C695TL79= FAE1-A 3920 silent C714T L86F FAE1-A 4489 Severe Missense G798AV114M FAE1-A 5495 Missense G801A A115T FAE1-A 3436 Missense G805A C116YFAE1-A 3533 Missense G810A D118N FAE1-A 3424 Missense G810A D118N FAE1-A5977 Missense C817T S120F FAE1-A 3821 Severe Missense C820T S121L FAE1-A4703 Missense G821A S121= FAE1-A 6126 silent G867A E137K FAE1-A 6361Missense G877A S140N FAE1-A 3284 Severe Missense G997A R180K FAE1-A 3390Missense G997A R180K FAE1-A 5346 Missense G1005A G183S FAE1-A 6655Severe Missense C1042T T195I FAE1-A 5557 Severe Missense G1061A M201IFAE1-A 4088 Severe Missense G1065A V203I FAE1-A 4469 Missense C1072TT205I FAE1-A 4500 Severe Missense C1083T R209* FAE1-A 3395 NonsenseC1091T N211= FAE1-A 5486 silent G1120A G221D FAE1-A 6386 Severe MissenseC1141T A228V FAE1-A 4467 Severe Missense C1167T H237Y FAE1-A 4164 SevereMissense C1167T H237Y FAE1-A 4318 Severe Missense G1254A V266I FAE1-A3365 Missense G1258A S267N FAE1-A 3783 Severe Missense C1272T R272CFAE1-A 5401 Severe Missense G1311A G285R FAE1-A 3799 Missense G1354AR299Q FAE1-A 5095 Severe Missense G1366A G303E FAE1-A 3820 SevereMissense G1387A R310Q FAE1-A 6528 Missense G1390A C311Y FAE1-A 3631Severe Missense G1401A G315R FAE1-A 4257 Missense G1402A G315E FAE1-A6186 Missense G1402A G315E FAE1-A 6446 Missense G1407A D317N FAE1-A 3897Severe Missense G1416A G320S FAE1-A 5197 Severe Missense G1426A G323EFAE1-A 5680 Severe Missense G1426A G323E FAE1-A 6284 Severe MissenseC1450T T331I FAE1-A 4412 Missense G1463A G335= FAE1-A 5117 silent G1518AE354K FAE1-A 3597 Severe Missense

TABLE 11 Summary of Camelina FAE1 B mutants Nucleotide Change EffectPrimer set PLANT ID Mutation Score C710T P76L FAE1B 5778 Severe MissenseC718T L79F FAE1B 5840 Severe Missense G724A D81N FAE1B 6324 SevereMissense C731T S83L FAE1B 4318 Severe Missense C817T R112W FAE1B 4140Severe Missense G823A V114M FAE1B 3768 Missense G823A V114M FAE1B 5966Missense C845T S121L FAE1B 3758 Missense G858A L125= FAE1B 3709 silentG887A G135D FAE1B 4015 Severe Missense C907T Q142* FAE1B 5951 NonsenseC928T P149S FAE1B 5107 Severe Missense C952T R157C FAE1B 4840 SevereMissense G953A R157H FAE1B 4239 Severe Missense G958A E159K FAE1B 6322Severe Missense G969A Q162= FAE1B 3529 silent G988A E169K FAE1B 3734Severe Missense C1019T P179L FAE1B 3873 Severe Missense G1031A G183DFAE1B 4135 Missense G1042A V187M FAE1B 6517 Severe Missense C1063T P194SFAE1B 6478 Severe Missense C1082T A200V FAE1B 3986 Severe MissenseG1086A M201I FAE1B 3895 Missense G1109A R209Q FAE1B 4139 Severe MissenseC1154T A224V FAE1B 3352 Severe Missense C1229T T249I FAE1B 4169 SevereMissense G1231A E250K FAE1B 6678 Severe Missense C1271T S263F FAE1B 3829Severe Missense C1271T S263F FAE1B 6700 Severe Missense G1275A M264IFAE1B 6308 Severe Missense G1306A G275R FAE1B 5333 Severe MissenseC1310T A276V FAE1B 3241 Severe Missense G1314A A277= FAE1B 4884 silentC1310T A276V FAE1B 3284 Severe Missense C1325T S281F FAE1B 5343 SevereMissense G1337A G285E FAE1B 3358 Missense G1337A G285E FAE1B 3821Missense G1343A R287Q FAE1B 5930 Silent C1352T S290F FAE1B 4882 SevereMissense C1384T H301Y FAE1B 4687 Severe Missense C1389T T302= FAE1B 5840silent G1412A R310Q FAE1B 3936 Missense G1417A V312M FAE1B 3173 SevereMissense G1427A G315E FAE1B 3926 Missense G1435A E318K FAE1B 6479Missense G1441A G320S FAE1B 3842 Severe Missense C1493T A337V FAE1B 4630Severe Missense C1522T P347S FAE1B 3912 Severe Missense

TABLE 12 Summary of Camelina FAE1 C mutants Nucleotide Change EffectPrimer set Plant ID Mutation Score A506T T15S FAE1-C 3688 Missense A506TT15S FAE1-C 4325 Missense A506T T15S FAE1-C 4907 Missense A506T TI5SFAE1-C 6025 Missense A506T TI5S FAE1-C 6695 Missense C564T S34F FAE1-C4965 Missense C605T L48F FAE1-C 6835 Missense G704A D81N FAE1-C 4510Severe Missense C719T L86F FAE1-C 5015 Severe Missense G798A R112QFAE1-C 4184 Missense C802T N113= FAE1-C 6130 silent C822T SI2OF FAE1-C3886 Severe Missense C825T 5121L FAE1-C 4255 Missense G840A R126K FAE1-C5936 Missense G855A R131H FAE1-C 3725 Severe Missense G855A R131H FAE1-C4813 Severe Missense C858T 5132L FAE1-C 5951 Severe Missense C887T P142SFAE1-C 3918 Missense C887T P142S FAE1-C 4198 Missense C906T P148L FAE1-C4068 Severe Missense C911T Q150* FAE1-C 5566 Nonsense C911T Q150* FAE1-C6139 Nonsense G926A A155T FAE1-C 3923 Missense G933A R157H FAE1-C 5576Severe Missense G982A E173= FAE1-C 3367 silent C987T T175I FAE1-C 3247Severe Missense G1010A G1835 FAE1-C 3365 Severe Missense C1047T T195IFAE1-C 5891 Severe Missense G1067A V202I FAE1-C 5975 Missense C1088TR209* FAE1-C 6476 Nonsense G1115A G218R FAE1-C 3970 Severe MissenseG1137A G225D FAE1-C 3911 Severe Missense C1154T L231F FAE1-C 6643 SevereMissense G1175A V238I FAE1-C 3380 Missense C1251T 5263F FAE1-C 5793Severe Missense C1252T S263= FAE1-C 3885 silent G1255A M264I FAE1-C 5422Severe Missense G1283A G274S FAE1-C 4945 Severe Missense G1287A G275EFAE1-C 3749 Severe Missense C1305T 5281F FAE1-C 3401 Severe MissenseC1305T 5281F FAE1-C 4608 Severe Missense G1316A G285R FAE1-C 4123Missense C1353T T297M FAE1-C 3427 Severe Missense G1359A R299Q FAE1-C3166 Severe Missense C1400T Q313* FAE1-C 5114 Nonsense C1403T Q314*FAE1-C 4162 Nonsense G1406A G315R FAE1-C 3776 Missense G1472A A337TFAE1-C 4852 Missense C1486T N341= FAE1-C 4399 silent C1494T T344M FAE1-C5013 Severe Missense C1502T P347S FAE1-C 6553 Severe Missense

As tables 7-12 indicate, multiple mutants were isolated in each FAD2 orFAE1 gene copy. The types of mutants include missense, severe missense,nonsense and silent mutations.

Example 12 Fatty Acids Composition in FAD2 and FAE1 Mutants

Fatty acid methyl ester (FAME) composition in Camelina FAD2 mutants wasanalyzed in a preliminary test by gas chromatography (GC) following theprotocol described in Example 1. The results were shown in Table 13.

TABLE 13 % FAME content in Camelina FAD2 mutants Cs 32 Combined FAD2AFAD2A missense FAD2A missense wild Null Q44* G150E S229F FAD2B W91*Mutation type Population HOMO HOMO NULL HOMO NULL HOMO NULL sample size10 14 8 7 4 5 4 6 6 C18:1 14.4 ± 0.4 14.3 ± 2.0 22.6 ± 1.2 24.0 ± 1.217.1 ± 0.9 19.2 ± 1.3 13.9 ± 0.9 18.4 ± 0.6 12.8 ± 0.5 C18:2 21.4 ± 0.828.8 ± 2.8 20.3 ± 1.1 19.0 ± 0.5 26.9 ± 1.2 22.2 ± 1.1 26.8 ± 1.4 28.7 ±0.4 31.5 ± 2.0 C18:3 33.7 ± 0.6 25.4 ± 1.9 26.2 ± 1.8 26.1 ± 1.6 26.0 ±1.1 25.7 ± 1.2 26.5 ± 1.3 23.8 ± 1.5 24.2 ± 2.2 C20:1 15.5 ± 1.0 10.7 ±1.4 12.2 ± 1.0 13.1 ± 1.4 10.5 ± 1.3 13.4 ± 1.0 10.8 ± 1.3 10.2 ± 1.610.7 ± 1.2 % increase in 56.9% 66.7% 33.3% 27.8% 18:1 relative to wildtype seeds Note: HOMO means the plants are all homozygous mutants at thespecified locus. NULL means there is no mutation at the specified locus.% means % of FAME composition

As the results indicate, an obvious increase of oleic acid (18:1) wasobserved in certain FAD2 mutants tested compared to NULL control plants.Thus, the data supports the inventors' prediction very well thatdisruption in one, two or more FAD2 gene in Camelina is sufficient toalter its fatty acid composition, and more specifically, to increase theoleic acid (18:1) concentration.

More mutants in FAD2 genes and FAE1 genes were subjected to GC analysis.To select mutants with potentially the most profound phenotype, FAD2 A,B, and C, or FAE1 A, B, and C protein sequences were analyzed againstorthologs in Arabidopsis, Crambe, B. rapa HEAC, B. rapa LEAC, meadowfoam, and nasturtium. It is preferred that a mutation happens at theposition which is conserved through reference species, and/or a positiondescribed before as conserved in orthologs or close-related genes inother species (e.g., see reference 52, Ghanevati and Jaworski, 2002, andJet et al., Dissection of malonyl-coenzyme A decarboxylation frompolyketide formation in the reaction mechanism of a plant polyketidesynthase, Biochemistry, 39:890-902). For example, the G150E, Q44*(nonsense), S229F and W91* (nonsense) mutations in FAD2 genes arepotentially very promising as are the following mutants in FAE1: Q142*(nonsense), R209* (nonsense), G221D and H301Y.

Two independent GC analyses of fatty acid compositions in FAD2 A andFAD2 B mutants were conducted, and the results are shown in Tables 14and 15. Mutants with clear increases in oleic acid were selected, andtheir results from both GC runs were averaged together to produce Table16. Some of these tested mutations have obvious increased oleic acid(18:1), such as FAD2 A mutants G150E, Q44*, S229F, and FAD2 B mutantsW91* compared to NULL population or wild-type Cs32 control plants, whileno significant difference was found between NULL population and wildtype Cs32 plants. Table 17 shows the fatty acid compositions of selectedFAD2 mutants for one of the independent GC analyses. The result of Table16 is further summarized in FIG. 12A. As the results indicate, thesemutants have evident increased oleic acid (18:1) and reducedpolyunsaturated fatty acids (e.g., 18:2 and 18:3) in seed oil, just asthe inventors predicted.

A third independent GC analysis was conducted in which FAD2 C mutantswere included. This was a preliminary analysis where seeds fromheterozygous plants were used, resulting in a mixed populationcontaining null, heterozygous and homozygous seeds. The results (seeTable 18 in U.S. Provisional Application No. 61/318,273, incorporated byreference in its entirety) showed that all tested FAD2 C mutants do nothave significant induction of 18:1 fatty acid, as compared to Cs32control plants. While not wishing to be bound by any particular theory,the results suggest that any potential increase in 18:1 in a FAD2 Cmutant plant is not detectable in progeny from heterozygous plants,where the mixture of wild type, heterozygous and homozygous seeds maydilute the effects of the homozygous seed.

The same preliminary third GC run analyzed mutants at the FAE1 loci.Though results (see Table 19 and Table 20 in U.S. ProvisionalApplication No. 61/318,273) showed that some of these tested mutants,for example FAE1 A mutant R272C, FAE1 B mutants S281F and R209Q, andFAE1C mutants Q313* and Q150* had obvious decreased 20:1 and/or 22:1 inseed oil relative to wild type Cs32 plants, the inclusion of asignificant number of heterozygous lines may have confounded the resultsas was the case with the FAD2 C results above.

A fourth independent GC analysis (results shown in Tables 18a and 19a ofthe present specification) was conducted on M4 or M5 generation FAD2 A,FAD2 B, FAD2 C, FAE1 A, FAE1 B and FAE1 C mutants. This analysisincluded multiple homozygous lines for a given FAD2 or FAE1 mutation,which conferred more confidence in the results due to multiple samplesfor a given mutation. In addition, the inventors limited the number ofheterozygous lines analyzed where the seeds were a mixture of homozygous(designated ‘hom’), heterozygous (designated ‘het’) and null because ofambiguous results in the third GC run. In test 4, Arabidopsis FAD2 andFAE1 mutants, wild type Camelina sativa CS32, and null sibling lines notcarrying a FAD2 or FAE1 mutation were included as controls. From thisanalysis, some FAD2 A Q44* and G150E, FAD2 B W91* and G150E, and FAD2CW87* homozygous or heterozygous lines clearly had greater 18:1 fattyacid levels compared to their null sibling lines or the CS32 control.

For FAE1, some FAE1 A G221D, FAE1 B Q142* and H301Y, and FAE1 C R209*homozygous or heterozygous lines clearly had lower 20:1 fatty acidlevels and/or lower 22:1 fatty acid levels compared to their nullsibling lines or the CS32 control. The FAE1 data from the fourth GC runis summarized in FIG. 12B (this Figure replaces FIG. 14B from U.S.Provisional Application No. 61/318,273, which summarized FAE1 data fromthe third GC run). This data supports the inventors' prediction thatdisruption in one, two or more FAE1 genes in Camelina is sufficient toalter its fatty acid composition, and more specifically, to decrease thevery long chain (for example 20:1 and 22:1) fatty acid content.

The fourth GC analysis did not include some FAD2 C, FAE1 A, FAE1 B andFAE1 C mutants included in the third GC analysis due to pursuance of aselect number of mutant lines in the breeding program for FAD2 (A, B andC) and FAE1 (A, B and C) mutants. In particular, FAD2 C mutants Q346*,G150R, R242H, G190R were included in the third but not the fourth GCanalysis. Similarly, FAE1 A mutants G183S, R272C, C311Y, FAE1 B mutantsP76L, L79F, R157H, R209Q, E250K, W91*, and FAE1 C mutants R157H, G225D,L231F, G274S, Q313*, Q314* were included in the third but not the fourthGC analysis. The FAE1 C Q150* mutant, which was analyzed in test 3 butnot test 4, will be tested for fatty acid composition in future GC runs.In test 3, homozygous FAE1 C Q150* mutant plants were used for analysis.According to the GC data in test 3, the fatty acids composition inhomozygous FAE1 C Q150* mutant is as following: C16:0, 6.19%; C18:0,2.74%; C18:1, 13.81%; C18:2, 24.05%; C20:0, 0.97%; C18:3, 33.28%; C20:1,14.25%; C20:2, 1.79%; C20:3, 0.70%; and C22:1, 2.23%.

TABLE 14 Fatty Acids Composition in FAD2 mutants, sorted by mutation,Test No. 1 muta- geno- Line Gene SNP tion Plant # type C16:0 C18:0 C18:1C18:2 C20:0 C18:3 C20:1 C20:2 C20:3 C22:1 2362 FAD2A 5 G150E 4 HOMO 7.9%4.3% 22.9% 19.1% 1.9% 28.1% 12.3% 0.8% 0.7% 2.1% 2362 FAD2A 5 G150E 5HOMO 7.8% 3.8% 23.0% 19.1% 1.8% 28.1% 13.1% 0.7% 0.6% 2.1% 2362 FAD2 A 5G150E 1 HET 7.3% 3.8% 22.3% 20.7% 2.1% 25.7% 14.3% 0.9% 0.6% 2.3% 2362FAD2 A 5 G150E 2 HET 11.0% 5.4% 13.6% 15.0% 2.8% 34.6% 12.9% 1.4% 0.8%2.4% 2362 FAD2A 5 G150E 11 Null 8.3% 5.2% 17.9% 25.3% 2.7% 27.4% 10.0%1.0% 0.5% 1.7% 2362 FAD2A 5 G150E 18 Null 9.0% 5.1% 17.8% 28.2% 3.2%24.9% 8.9% 1.2% 0.6% 1.1% 2510 FAD2A 4 S147F 3 HOMO 7.2% 5.1% 15.9%24.4% 3.8% 28.4% 10.3% 1.3% 0.8% 2.7% 2510 FAD2A 4 S147F 10 HOMO 9.0%6.4% 15.9% 29.2% 3.5% 25.0% 7.1% 1.2% 0.6% 2.1% 2510 FAD2A 4 S147F 1 HET8.2% 5.5% 15.1% 27.2% 3.3% 27.9% 8.0% 1.3% 0.8% 2.8% 2510 FAD2A 4 S147F4 HET 8.4% 5.7% 15.8% 30.1% 3.7% 23.7% 8.5% 1.3% 0.3% 2.4% 2510 FAD2A 4S147F 2 Null 8.5% 4.7% 12.6% 28.2% 3.5% 27.7% 9.2% 1.7% 0.9% 2.9% 2510FAD2A 4 S147F 5 Null 7.7% 3.5% 14.0% 26.9% 2.5% 29.3% 10.7% 1.5% 0.9%3.0% 2579 FAD2A 7 S229F 1 HOMO 7.3% 3.4% 19.3% 23.6% 1.7% 26.5% 13.0%1.2% 0.7% 3.2% 2579 FAD2A 7 S229F 5 HOMO 8.0% 4.4% 20.9% 22.3% 2.2%27.4% 10.6% 1.1% 0.4% 2.8% 2579 FAD2A 7 S229F 2 HET 7.5% 3.6% 17.0%25.8% 0.1% 31.7% 10.1% 1.6% 0.4% 2.3% 2579 FAD2A 7 S229F 3 HET 7.7% 4.6%16.5% 25.0% 0.1% 30.5% 11.2% 1.3% 0.8% 2.4% 2579 FAD2A 7 S229F 10 Null8.4% 3.6% 12.0% 26.8% 2.6% 28.9% 11.9% 1.9% 0.9% 3.1% 2579 FAD2A 7 S229F12 Null 8.0% 5.2% 14.4% 26.1% 3.5% 28.9% 8.9% 1.5% 0.8% 2.8% 2764 FAD2A3 R144H 5 HOMO 7.5% 3.8% 22.1% 26.3% 0.1% 26.0% 10.5% 1.1% 0.5% 2.1%2764 FAD2A 3 R144H 8 HOMO 7.5% 3.3% 17.0% 21.6% 0.1% 34.4% 11.3% 1.2%0.9% 2.9% 2764 FAD2A 3 R144H 10 HOMO 6.9% 4.3% 17.6% 22.5% 2.9% 28.4%12.3% 1.2% 0.8% 3.2% 2764 FAD2A 3 R144H 16 HOMO 6.5% 3.0% 18.7% 24.5%1.7% 26.9% 13.3% 1.3% 0.7% 3.4% 2764 FAD2A 3 R144H 1 HET 6.9% 3.4% 18.4%24.5% 1.5% 27.7% 12.7% 1.3% 0.5% 3.0% 2764 FAD2A 3 R144H 2 HET 7.7% 3.9%15.7% 25.5% 0.1% 32.5% 10.1% 1.4% 0.8% 2.3% 2764 FAD2A 3 R144H 3 HET7.3% 3.5% 17.6% 26.5% 0.1% 29.1% 11.4% 1.4% 0.7% 2.4% 2764 FAD2A 3 R144H6 HET 7.7% 3.3% 16.8% 22.3% 0.1% 34.5% 10.8% 1.1% 0.9% 2.6% 2764 FAD2A 3R144H 4 Null 6.8% 3.5% 17.1% 27.8% 1.7% 24.9% 12.8% 1.5% 0.7% 3.1% 2764FAD2A 3 R144H 7 Null 7.1% 3.2% 17.1% 25.2% 1.5% 28.2% 12.5% 1.5% 0.5%3.3% 2785 FAD2C 11 H316Y 3 HOMO 8.1% 3.7% 16.6% 20.5% 2.6% 32.1% 11.2%1.3% 0.8% 3.2% 2785 FAD2C 11 H316Y 10 HOMO 8.3% 3.7% 15.2% 16.7% 2.3%34.2% 14.3% 1.2% 1.1% 3.1% 2785 FAD2C 11 H316Y 12 HOMO 8.5% 3.5% 14.3%18.0% 2.7% 32.8% 14.1% 1.2% 1.2% 3.7% 2785 FAD2C 11 H316Y 18 HOMO 8.1%3.9% 14.8% 19.1% 2.1% 35.0% 11.0% 1.3% 1.3% 3.4% 2785 FAD2C 11 H316Y 1HET 8.8% 4.0% 14.3% 22.0% 2.0% 33.3% 9.9% 1.6% 0.8% 3.4% 2785 FAD2C 11H316Y 6 HET 8.9% 3.9% 14.4% 20.5% 0.1% 36.0% 11.1% 1.3% 1.1% 2.8% 2785FAD2C 11 H316Y 7 HET 8.2% 3.9% 14.4% 20.5% 0.1% 36.1% 11.1% 1.3% 1.1%3.1% 2785 FAD2C 11 H316Y 8 HET 9.0% 3.9% 12.6% 21.0% 0.1% 38.2% 10.2%1.5% 1.0% 2.5% 2785 FAD2C 11 H316Y 2 Null 8.6% 3.8% 11.3% 20.6% 2.8%35.1% 11.1% 1.6% 1.4% 3.7% 2785 FAD2C 11 H316Y 5 Null 8.8% 4.7% 12.8%20.7% 3.0% 34.2% 10.3% 1.4% 1.1% 3.0% 2812 FAD2B 9 H145Y 1 HOMO 8.3%4.1% 15.1% 27.4% 2.9% 24.6% 12.4% 1.5% 0.7% 3.1% 2812 FAD2B 9 H145Y 2HOMO 9.9% 3.6% 13.3% 28.7% 2.4% 27.0% 9.3% 1.5% 0.8% 3.5% 2812 FAD2B 9H145Y 12 HET 7.8% 3.9% 16.6% 29.1% 2.6% 24.6% 10.7% 1.5% 0.7% 2.6% 2812FAD2B 9 H145Y 25 HET 7.8% 4.1% 15.9% 30.5% 2.2% 25.8% 9.0% 1.6% 0.7%2.4% 2826 FAD2A 2 Q44* 1 HOMO 7.7% 4.9% 22.2% 20.1% 2.1% 28.4% 11.0%0.8% 0.6% 2.1% 2826 FAD2A 2 Q44* 2 HOMO 7.8% 4.9% 19.9% 19.2% 2.6% 29.5%11.7% 1.0% 0.8% 2.6% 2826 FAD2A 2 Q44* 3 HOMO 7.5% 4.6% 22.9% 19.8% 2.1%27.7% 11.7% 0.8% 0.6% 2.3% 2826 FAD2A 2 Q44* 4 HOMO 7.7% 5.4% 24.7%20.3% 2.5% 25.7% 10.6% 0.7% 0.5% 1.9% 2826 FAD2A 2 Q44* 5 HOMO 7.6% 5.2%22.9% 19.1% 2.2% 28.1% 11.5% 0.8% 0.6% 2.0% 2826 FAD2A 2 Q44* 37 HET7.3% 4.8% 22.9% 19.1% 2.0% 28.5% 12.2% 0.7% 0.6% 1.9% 3006 FAD2B 8 W91*1 HOMO 7.9% 4.6% 18.9% 28.5% 1.7% 26.8% 8.9% 1.0% 0.5% 1.1% 3006 FAD2B 8W91* 2 HOMO 8.2% 5.3% 18.8% 29.2% 1.7% 26.3% 7.8% 0.9% 0.4% 1.3% 3006FAD2B 8 W91* 3 HOMO 8.0% 5.5% 18.4% 28.9% 2.7% 24.4% 8.6% 1.1% 0.5% 1.8%3006 FAD2B 8 W91* 4 HOMO 7.5% 5.1% 18.0% 28.9% 2.9% 22.9% 10.6% 1.3%0.5% 2.3% 3006 FAD2B 8 W91* 5 Null 7.8% 4.2% 12.3% 33.5% 2.9% 23.8% 9.3%2.1% 0.7% 3.5% 3006 FAD2B 8 W91* 7 Null 7.9% 4.4% 17.6% 30.0% 2.1% 24.9%9.7% 1.2% 0.5% 1.6% 3006 FAD2B 8 W91* 8 HET 8.0% 3.9% 13.4% 30.6% 2.2%26.9% 9.3% 2.0% 0.5% 3.1% Note: *stands for nonsense mutation; HOMOmeans the plants are all homozygous mutants at the specified locus. HETmeans the plants are heterozygous mutants at the specified locus. NULLmeans there is no mutation at the specified locus. % means % of FAMEcomposition

TABLE 15 Fatty Acids Composition in FAD2 mutants, sorted by mutation,Test No. 2 muta- Plant geno- # of Gene SNP tion # type samples C16:0C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C20:2 C20:3 C22:1 none none 1 CS3210  5.8% 2.3% 14.9% 20.3% 34.0% 1.2% 16.2% 1.8% 0.9% 2.7% controls nonenone 2 CS32 controls 6.1% 2.3% 14.6% 21.3% 33.3% 1.2% 15.6% 1.8% 1.2%2.6% none none 3 CS32 controls 6.2% 2.3% 14.1% 21.7% 34.2% 1.1% 15.5%1.8% 1.0% 2.1% none none 4 CS32 controls 6.0% 2.4% 14.2% 20.8% 34.5%1.2% 15.8% 1.6% 1.0% 2.4% none none 5 CS32 controls 6.7% 2.6% 14.6%23.1% 33.5% 0.7% 12.9% 1.8% 1.3% 2.7% none none 6 CS32 controls 6.2%2.5% 14.6% 21.6% 33.4% 1.0% 15.4% 1.8% 1.1% 2.5% none none 7 CS32controls 5.9% 2.4% 14.6% 22.2% 32.7% 1.3% 15.7% 1.8% 1.0% 2.5% none none8 CS32 controls 6.0% 2.3% 13.6% 21.0% 34.3% 1.4% 16.0% 1.9% 1.0% 2.6%none none 9 CS32 controls 5.9% 2.4% 14.3% 20.6% 33.6% 1.3% 16.2% 1.9%1.1% 2.7% none none 10 CS32 controls 6.2% 2.4% 13.9% 21.4% 33.2% 1.3%15.9% 1.9% 1.2% 2.6% FAD2B/ 51 D60N Y1 605 1 8.7% 2.5% 10.4% 29.0% 28.4%1.7% 13.3% 2.1% 0.9% 3.0% C FAD2A 5 G150E 4 HOMO 1 8.0% 4.4% 22.5% 18.8%27.5% 2.2% 12.7% 0.5% 1.7% 1.7% FAD2A 5 G150E 5 HOMO 1 7.8% 4.2% 23.2%18.4% 26.6% 2.2% 14.2% 0.7% 0.6% 2.2% FAD2A G150E 20 Null 2 new 8.7%4.2% 16.2% 26.7% 25.7% 2.2% 12.0% 1.3% 0.4% 2.4% FAD2A G150E 24 Null8.7% 5.1% 16.4% 27.4% 26.0% 2.2% 10.9% 1.1% 0.6% 1.6% FAD2A G150E 6 HOMO5 new 7.7% 4.2% 26.1% 18.8% 25.2% 1.9% 13.3% 0.6% 0.5% 1.7% FAD2A G150E8 HOMO 8.1% 4.6% 24.2% 18.7% 28.1% 2.2% 10.2% 0.7% 0.5% 2.7% FAD2A G150E14 HOMO 8.2% 4.5% 24.6% 19.4% 24.7% 2.0% 13.7% 0.6% 0.3% 1.9% FAD2AG150E 23 HOMO 8.6% 4.7% 23.0% 19.0% 26.7% 2.1% 13.1% 0.6% 0.5% 1.8%FAD2A G150E 25 HOMO 8.0% 4.0% 24.2% 20.0% 23.7% 1.8% 14.6% 0.7% 0.5%2.5% FAD2A G150E 3 Het 3 new 7.9% 4.4% 21.1% 22.2% 27.2% 2.0% 12.4% 0.8%0.3% 1.7% at least FAD2A G150E 7 Het 8.5% 4.5% 19.1% 24.3% 24.8% 2.1%12.8% 1.0% 0.6% 2.2% FAD2A G150E 9 Het 8.4% 4.7% 17.9% 23.3% 27.5% 2.5%12.2% 1.0% 0.6% 1.8% FAD2A 2 Q44* 2 HOMO 1 8.3% 5.1% 22.8% 20.8% 26.7%2.0% 11.1% 0.8% 0.4% 2.0% FAD2A 2 Q44* 1 HOMO 1 7.8% 4.9% 19.7% 18.9%28.3% 2.6% 13.9% 0.9% 0.7% 2.4% FAD2A 2 Q44* 4 HOMO 1 7.6% 5.1% 23.7%20.2% 25.7% 2.4% 12.8% 0.6% 0.4% 1.5% FAD2A Q44* 40 Null 1 8.2% 6.1%25.6% 20.4% 24.3% 2.4% 10.8% 0.6% 0.3% 1.4% FAD2A Q44* 36 HOMO 3 7.8%5.5% 23.2% 21.4% 23.8% 2.5% 13.0% 0.7% 0.2% 1.8% FAD2A Q44* 38 HOMO 8.0%6.2% 22.3% 20.4% 25.4% 2.9% 12.4% 0.7% 0.3% 1.5% FAD2A Q44* 39 HOMO 9.2%5.6% 22.9% 22.0% 23.7% 2.3% 11.0% 0.7% 0.4% 2.0% FAD2A 3 R144H 16 HOMO 16.9% 2.9% 18.5% 26.2% 26.5% 1.6% 13.1% 1.2% 0.3% 2.9% FAD2A 3 R144H 5HOMO 1 7.1% 4.0% 21.7% 26.0% 23.2% 2.4% 12.1% 0.9% 0.3% 2.3% FAD2A R144H19 Null 2 (34) 7.1% 3.0% 17.8% 27.1% 26.0% 1.7% 12.8% 1.3% 0.4% 2.7%FAD2A R144H 25 Null 7.4% 4.3% 15.2% 25.6% 29.6% 2.4% 11.5% 1.2% 0.4%2.4% FAD2A 7 S229F 5 HOMO 1 7.6% 3.8% 19.6% 21.4% 27.4% 2.0% 13.9% 1.0%0.6% 2.6% FAD2A 7 S229F 3 HET 1 7.9% 5.1% 18.2% 26.0% 25.7% 2.0% 10.4%1.2% 0.7% 2.6% FAD2A 7 S229F 12 Null 1 8.6% 5.6% 15.0% 27.8% 26.6% 3.4%8.2% 1.4% 0.7% 2.8% FAD2A 7 S229F 10 Null 1 9.3% 5.7% 12.8% 28.1% 25.4%3.7% 10.1% 1.8% 0.4% 2.7% FAD2A S229F 7 HOMO 3 8.4% 5.8% 18.4% 22.7%24.5% 3.5% 11.8% 1.1% 0.6% 3.2% FAD2A S229F 9 HOMO 7.3% 5.1% 17.8% 22.4%25.2% 3.6% 14.0% 1.1% 0.6% 2.9% FAD2A S229F 19 HOMO 6.9% 5.0% 21.0%20.9% 24.8% 2.7% 14.3% 0.9% 0.6% 2.9% FAD2A S229F 13 Null 2 7.8% 5.6%14.1% 26.4% 25.6% 3.6% 12.2% 1.5% 0.7% 2.5% FAD2A S229F 14 Null 7.6%4.8% 13.8% 25.0% 28.3% 3.2% 12.6% 1.2% 0.8% 2.6% FAD2A S229F 4 Het 3(46) 7.6% 3.5% 15.6% 23.8% 27.3% 2.3% 14.2% 1.6% 0.8% 3.2% FAD2A S229F 6Het 7.7% 2.8% 14.8% 24.5% 29.4% 1.9% 13.5% 1.5% 0.8% 3.1% FAD2A S229F 8Het 7.3% 5.0% 14.2% 22.6% 28.2% 3.7% 13.5% 1.4% 1.0% 3.2% FAD2A S229F Y1610 7 5.3% 2.3% 16.5% 21.8% 29.8% 1.5% 16.2% 2.0% 1.2% 3.4% FAD2A S229FY2 610 5.5% 2.3% 16.1% 22.0% 30.6% 1.2% 16.2% 1.9% 1.2% 2.9% FAD2A S229FY3 610 6.5% 2.2% 17.0% 23.3% 29.5% 1.1% 15.3% 1.7% 0.9% 2.4% FAD2A S229FY4 610 5.9% 2.0% 14.8% 21.8% 34.2% 0.9% 14.7% 2.0% 1.1% 2.5% FAD2A S229FY5 610 5.9% 2.0% 14.4% 22.3% 34.7% 1.0% 14.7% 1.8% 1.0% 2.3% FAD2A S229FY6 610 5.5% 2.2% 16.7% 21.6% 31.9% 1.2% 15.2% 1.8% 1.1% 2.8% FAD2A S229FY7 610 6.3% 2.5% 17.8% 23.7% 28.4% 1.4% 14.8% 1.6% 0.9% 2.6% FAD2B W91*1 HOMO 1 7.7% 5.1% 19.4% 28.6% 23.9% 1.9% 10.9% 0.9% 0.4% 1.2% FAD2BW91* 8 HET 1 7.8% 5.5% 18.5% 27.5% 24.4% 2.5% 10.9% 0.9% 0.5% 1.5% FAD2BW91* 7 Null 1 7.6% 3.9% 12.4% 32.4% 23.9% 2.6% 11.5% 1.9% 0.7% 3.1%FAD2B W91* 5 Null 1 7.9% 4.2% 12.8% 29.4% 26.1% 2.6% 11.8% 1.7% 0.7%2.9% FAD2B W91* 10 Null 4 8.4% 5.0% 12.1% 28.8% 26.9% 2.8% 11.4% 1.7%0.8% 2.0% FAD2B W91* 13 Null 7.6% 4.8% 13.0% 33.7% 21.9% 2.8% 10.8% 1.8%0.6% 2.9% FAD2B W91* 23 Null 8.3% 5.8% 13.4% 31.9% 21.4% 4.0% 10.6% 1.7%0.4% 2.6% FAD2B W91* 24 Null 9.1% 5.1% 13.1% 32.8% 24.8% 2.2% 8.4% 1.8%0.3% 2.5% FAD2B W91* 21 HOMO 2 7.9% 5.0% 17.9% 28.5% 23.5% 2.2% 11.5%1.1% 0.5% 1.8% FAD2B W91* 22 HOMO 7.7% 6.0% 18.1% 28.1% 21.8% 3.3% 11.5%1.1% 0.4% 2.0% FAD2B W91* Y1 1105 9 5.9% 2.5% 19.7% 22.9% 33.6% 0.6%12.3% 1.0% 0.7% 0.8% FAD2B W91* Y2 1105 6.4% 2.5% 19.0% 24.5% 30.4% 1.0%13.0% 1.2% 0.7% 1.3% FAD2B W91* Y3 1105 6.8% 2.7% 18.8% 25.4% 31.6% 0.6%11.5% 1.2% 0.5% 1.0% FAD2B W91* Y4 1105 7.1% 3.0% 19.8% 26.8% 28.0% 1.0%11.9% 1.0% 0.5% 0.9% FAD2B W91* Y5 1105 6.4% 2.4% 19.8% 25.1% 29.7% 0.8%12.7% 1.1% 0.6% 1.5% FAD2B W91* Y6 1105 6.8% 2.6% 18.1% 26.1% 29.6% 1.1%12.7% 1.3% 0.5% 1.3% FAD2B W91* Y7 1105 6.0% 2.7% 14.6% 24.5% 30.3% 1.0%14.9% 2.0% 1.1% 2.9% FAD2B W91* Y8 1105 5.9% 2.5% 19.0% 23.0% 32.0% 0.9%13.7% 1.2% 0.5% 1.2% FAD2B W91* Y9 1105 6.5% 2.5% 17.2% 24.2% 31.7% 1.1%13.4% 1.1% 0.7% 1.5% Note: *stands for nonsense mutation; HOMO means theplants are all homozygous mutants at the specified locus. HET means theplants are heterozygous mutants at the specified locus. NULL means thereis no mutation at the specified locus. % means % of FAME composition

TABLE 16 Fatty Acids Composition in selected FAD2 mutants, sorted bymutation, Average of Test No. 1 and Test No. 2 muta- Plant # of Gene SNPtion # genotype samples C16:0 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C20:2C20:3 C22:1 none none CS32 1 CS32 10  5.79% 2.28% 14.91% 20.26% 34.01%1.20% 16.16% 1.75% 0.92% 2.71% controls controls none none CS32 2 CS326.07% 2.30% 14.63% 21.35% 33.27% 1.19% 15.64% 1.80% 1.17% 2.56% controlscontrols none none CS32 3 CS32 6.17% 2.30% 14.13% 21.74% 34.22% 1.08%15.46% 1.79% 1.02% 2.09% controls controls none none CS32 4 CS32 6.03%2.40% 14.23% 20.78% 34.53% 1.23% 15.81% 1.56% 1.01% 2.41% controlscontrols none none CS32 5 CS32 6.73% 2.57% 14.63% 23.08% 33.52% 0.75%12.87% 1.83% 1.34% 2.67% controls controls none none CS32 6 CS32 6.22%2.46% 14.57% 21.59% 33.42% 0.98% 15.40% 1.84% 1.05% 2.47% controlscontrols none none CS32 7 CS32 5.88% 2.42% 14.58% 22.23% 32.70% 1.26%15.70% 1.79% 0.97% 2.46% controls controls none none CS32 8 CS32 6.03%2.26% 13.61% 20.96% 34.27% 1.35% 15.97% 1.89% 1.04% 2.62% controlscontrols none none CS32 9 CS32 5.93% 2.38% 14.34% 20.60% 33.61% 1.26%16.23% 1.85% 1.11% 2.70% controls controls none none CS32 10 CS32 6.19%2.42% 13.93% 21.40% 33.20% 1.35% 15.85% 1.88% 1.20% 2.57% controlscontrols FAD2A Cs32 14.36% 21.40% 33.67% 15.51% AVE FAD2A Cs32 SD 0.39%0.83% 0.57% 0.96% FAD2A 5 G150E 4 HOMO 1 7.98% 4.39% 22.51% 18.83%27.52% 2.20% 12.69% 0.47% 1.71% 1.71% FAD2A 5 G150E 5 HOMO 1 7.80% 4.19%23.22% 18.43% 26.56% 2.16% 14.17% 0.68% 0.56% 2.24% FAD2A G150E 6 HOMO 5new 7.68% 4.22% 26.13% 18.78% 25.20% 1.94% 13.30% 0.55% 0.46% 1.74%FAD2A G150E 8 HOMO 8.09% 4.58% 24.20% 18.71% 28.10% 2.24% 10.23% 0.73%0.46% 2.66% FAD2A G150E 14 HOMO 8.24% 4.46% 24.63% 19.43% 24.68% 2.02%13.72% 0.64% 0.29% 1.89% FAD2A G150E 23 HOMO 8.60% 4.73% 22.96% 19.00%26.72% 2.07% 13.07% 0.58% 0.52% 1.76% FAD2A G150E 25 HOMO 8.05% 4.01%24.16% 19.98% 23.65% 1.84% 14.60% 0.72% 0.52% 2.46% FAD2A G150E Homo23.97% 19.02% 26.06% 13.11% AVE FAD2A G150E Homo SD 1.22% 0.52% 1.60%1.43% FAD2A G150E 20 Null 2 new 8.73% 4.20% 16.24% 26.75% 25.73% 2.17%12.03% 1.30% 0.44% 2.41% FAD2A 5 G150E 11 Null 8.29% 5.23% 17.87% 25.33%27.41% 2.72% 9.99% 0.98% 0.49% 1.69% FAD2A 5 G150E 18 Null 8.98% 5.13%17.82% 28.18% 24.86% 3.19% 8.92% 1.20% 0.57% 1.14% FAD2A G150E 24 Null8.74% 5.08% 16.38% 27.40% 26.04% 2.17% 10.88% 1.09% 0.63% 1.59% FAD2AG150E Null AVE 17.08% 26.91% 26.01% 10.46% FAD2A G150E null SD 0.89%1.21% 1.06% 1.32% FAD2A 2 Q44* 2 HOMO 1 8.29% 5.10% 22.83% 20.76% 26.73%2.03% 11.13% 0.78% 0.39% 1.96% FAD2A 2 Q44* 1 HOMO 1 7.80% 4.89% 19.67%18.86% 28.30% 2.61% 13.90% 0.87% 0.73% 2.36% FAD2A 2 Q44* 4 HOMO 1 7.63%5.05% 23.75% 20.19% 25.73% 2.39% 12.83% 0.60% 0.38% 1.45% FAD2A 2 Q44* 5HOMO 7.61% 5.25% 22.91% 19.10% 28.07% 2.20% 11.49% 0.78% 0.62% 1.95%FAD2A 2 Q44* 3 HOMO 7.50% 4.63% 22.86% 19.84% 27.72% 2.09% 11.66% 0.80%0.63% 2.26% FAD2A Q44* 36 HOMO 3 7.79% 5.55% 23.24% 21.36% 23.80% 2.46%13.03% 0.71% 0.22% 1.84% FAD2A Q44* 38 HOMO 7.98% 6.21% 22.35% 20.37%25.41% 2.87% 12.40% 0.66% 0.28% 1.47% FAD2A Q44* 39 HOMO 9.19% 5.64%22.93% 22.02% 23.75% 2.34% 10.96% 0.74% 0.42% 2.00% FAD2A Q44* Homo22.57% 20.31% 26.19% 12.18% AVE FAD2A Q44* homo SD 1.24% 1.07% 1.82%1.04% FAD2A 7 S229F 5 HOMO 1 7.60% 3.79% 19.62% 21.37% 27.42% 2.01%13.94% 0.97% 0.63% 2.64% FAD2A 7 S229F 1 HOMO 7.31% 3.36% 19.34% 23.61%26.47% 1.74% 13.03% 1.25% 0.73% 3.17% FAD2A S229F 7 HOMO 3 8.44% 5.78%18.39% 22.68% 24.53% 3.52% 11.78% 1.09% 0.56% 3.22% FAD2A S229F 9 HOMO7.32% 5.15% 17.75% 22.35% 25.15% 3.56% 14.04% 1.11% 0.65% 2.91% FAD2AS229F 19 HOMO 6.92% 4.96% 21.02% 20.89% 24.77% 2.73% 14.28% 0.92% 0.63%2.87% FAD2A S229F 20 HOMO 19.23% 22.18% 25.67% 13.42% AVE FAD2A S229F 21HOMO 1.25% 1.08% 1.23% 1.03% SD FAD2A 7 S229F 12 Null 1 8.62% 5.59%14.98% 27.76% 26.56% 3.41% 8.20% 1.36% 0.69% 2.82% FAD2A 7 S229F 10 Null1 9.35% 5.67% 12.84% 28.10% 25.37% 3.68% 10.10% 1.77% 0.41% 2.71% FAD2AS229F 13 Null 2 7.77% 5.64% 14.08% 26.36% 25.64% 3.59% 12.18% 1.49%0.75% 2.51% FAD2A S229F 14 Null 7.57% 4.84% 13.78% 24.96% 28.33% 3.24%12.64% 1.21% 0.85% 2.59% FAD2A S229F 15 Null AVE 13.92% 26.80% 26.47%10.78% FAD2A S229F 16 Null SD 0.88% 1.44% 1.34% 2.05% FAD2B W91* 1 HOMO1 7.72% 5.07% 19.39% 28.57% 23.91% 1.93% 10.91% 0.92% 0.44% 1.15% FAD2BW91* 21 HOMO 2 7.85% 5.02% 17.95% 28.51% 23.54% 2.22% 11.50% 1.10% 0.53%1.78% FAD2B W91* 22 HOMO 7.73% 5.99% 18.08% 28.11% 21.75% 3.34% 11.49%1.09% 0.44% 1.97% FAD2B 8 W91* 2 HOMO 8.23% 5.35% 18.80% 29.20% 26.29%1.68% 7.84% 0.86% 0.41% 1.34% FAD2B 8 W91* 3 HOMO 8.05% 5.50% 18.37%28.87% 24.40% 2.69% 8.63% 1.15% 0.53% 1.82% FAD2B 8 W91* 4 HOMO 7.54%5.09% 17.96% 28.89% 22.95% 2.92% 10.62% 1.25% 0.51% 2.27% FAD2B W91* 5HOMO 18.43% 28.69% 23.80% 10.17% AVE FAD2B W91* 6 HOMO 0.57% 0.38% 1.52%1.55% SD FAD2B W91* 7 Null 1 7.61% 3.94% 12.42% 32.37% 23.94% 2.61%11.48% 1.87% 0.68% 3.09% FAD2B W91* 5 Null 1 7.93% 4.16% 12.77% 29.39%26.08% 2.58% 11.78% 1.68% 0.74% 2.89% FAD2B W91* 10 Null 4 8.35% 5.04%12.13% 28.77% 26.89% 2.84% 11.44% 1.69% 0.82% 2.04% FAD2B W91* 13 Null7.61% 4.83% 12.99% 33.71% 21.94% 2.81% 10.77% 1.84% 0.62% 2.88% FAD2BW91* 23 Null 8.31% 5.84% 13.38% 31.92% 21.35% 4.00% 10.55% 1.66% 0.36%2.62% FAD2B W91* 24 Null 9.08% 5.10% 13.10% 32.82% 24.75% 2.16% 8.39%1.77% 0.34% 2.49% FAD2B W91* 25 Null AVE 12.80% 31.50% 24.16% 10.73%FAD2B W91* 26 Null SD 0.46% 1.97% 2.21% 1.24% Note: *stands for nonsensemutation; HOMO means the plants are all homozygous mutants at thespecified locus. HET means the plants are heterozygous mutants at thespecified locus. NULL means there is no mutation at the specified locus.% means % of FAME composition

TABLE 17 Fatty Acids Composition in selected FAD2 mutants, sorted bymutation, Test 2 muta- Plant # of Gene SNP tion # genotype samples C16:0C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C20:2 C20:3 C22:1 none none CS32 1CS32 10  5.8% 2.3% 14.9% 20.3% 34.0% 1.2% 16.2% 1.8% 0.9% 2.7% controlscontrols none none CS32 2 CS32 controls 6.1% 2.3% 14.6% 21.3% 33.3% 1.2%15.6% 1.8% 1.2% 2.6% controls none none CS32 3 CS32 controls 6.2% 2.3%14.1% 21.7% 34.2% 1.1% 15.5% 1.8% 1.0% 2.1% controls none none CS32 4CS32 controls 6.0% 2.4% 14.2% 20.8% 34.5% 1.2% 15.8% 1.6% 1.0% 2.4%controls none none CS32 5 CS32 controls 6.7% 2.6% 14.6% 23.1% 33.5% 0.7%12.9% 1.8% 1.3% 2.7% controls none none CS32 6 CS32 controls 6.2% 2.5%14.6% 21.6% 33.4% 1.0% 15.4% 1.8% 1.1% 2.5% controls none none CS32 7CS32 controls 5.9% 2.4% 14.6% 22.2% 32.7% 1.3% 15.7% 1.8% 1.0% 2.5%controls none none CS32 8 CS32 controls 6.0% 2.3% 13.6% 21.0% 34.3% 1.4%16.0% 1.9% 1.0% 2.6% controls none none CS32 9 CS32 controls 5.9% 2.4%14.3% 20.6% 33.6% 1.3% 16.2% 1.9% 1.1% 2.7% controls none none CS32 10CS32 controls 6.2% 2.4% 13.9% 21.4% 33.2% 1.3% 15.9% 1.9% 1.2% 2.6%controls FAD2A 5 G150E 4 HOMO 1 8.0% 4.4% 22.5% 18.8% 27.5% 2.2% 12.7%0.5% 1.7% 1.7% FAD2A 5 G150E 5 HOMO 1 7.8% 4.2% 23.2% 18.4% 26.6% 2.2%14.2% 0.7% 0.6% 2.2% FAD2A G150E 6 HOMO 5 new 7.7% 4.2% 26.1% 18.8%25.2% 1.9% 13.3% 0.6% 0.5% 1.7% FAD2A G150E 8 HOMO 8.1% 4.6% 24.2% 18.7%28.1% 2.2% 10.2% 0.7% 0.5% 2.7% FAD2A G150E 14 HOMO 8.2% 4.5% 24.6%19.4% 24.7% 2.0% 13.7% 0.6% 0.3% 1.9% FAD2A G150E 23 HOMO 8.6% 4.7%23.0% 19.0% 26.7% 2.1% 13.1% 0.6% 0.5% 1.8% FAD2A G150E 25 HOMO 8.0%4.0% 24.2% 20.0% 23.7% 1.8% 14.6% 0.7% 0.5% 2.5% FAD2A 2 Q44* 2 HOMO 18.3% 5.1% 22.8% 20.8% 26.7% 2.0% 11.1% 0.8% 0.4% 2.0% FAD2A 2 Q44* 1HOMO 1 7.8% 4.9% 19.7% 18.9% 28.3% 2.6% 13.9% 0.9% 0.7% 2.4% FAD2A 2Q44* 4 HOMO 1 7.6% 5.1% 23.7% 20.2% 25.7% 2.4% 12.8% 0.6% 0.4% 1.5%FAD2A 2 Q44* 5 HOMO FAD2A 2 Q44* 3 HOMO FAD2A Q44* 36 HOMO 3 7.8% 5.5%23.2% 21.4% 23.8% 2.5% 13.0% 0.7% 0.2% 1.8% FAD2A Q44* 38 HOMO 8.0% 6.2%22.3% 20.4% 25.4% 2.9% 12.4% 0.7% 0.3% 1.5% FAD2A Q44* 39 HOMO 9.2% 5.6%22.9% 22.0% 23.7% 2.3% 11.0% 0.7% 0.4% 2.0% FAD2A 7 S229F 5 HOMO 1 7.6%3.8% 19.6% 21.4% 27.4% 2.0% 13.9% 1.0% 0.6% 2.6% FAD2A 7 S229F 1 HOMOFAD2A S229F 7 HOMO 3 8.4% 5.8% 18.4% 22.7% 24.5% 3.5% 11.8% 1.1% 0.6%3.2% FAD2A S229F 9 HOMO 7.3% 5.1% 17.8% 22.4% 25.2% 3.6% 14.0% 1.1% 0.6%2.9% FAD2A S229F 19 HOMO 6.9% 5.0% 21.0% 20.9% 24.8% 2.7% 14.3% 0.9%0.6% 2.9% FAD2B W91* 1 HOMO 1 7.7% 5.1% 19.4% 28.6% 23.9% 1.9% 10.9%0.9% 0.4% 1.2% FAD2B W91* 21 HOMO 2 7.9% 5.0% 17.9% 28.5% 23.5% 2.2%11.5% 1.1% 0.5% 1.8% FAD2B W91* 22 HOMO 7.7% 6.0% 18.1% 28.1% 21.8% 3.3%11.5% 1.1% 0.4% 2.0% FAD2B 8 W91* 2 HOMO FAD2B 8 W91* 3 HOMO FAD2B 8W91* 4 HOMO FAD2A G150E 20 Null 2 8.7% 4.2% 16.2% 26.7% 25.7% 2.2% 12.0%1.3% 0.4% 2.4% FAD2A 5 G150E 11 Null 8.3% 5.2% 17.9% 25.3% 27.4% 2.7%10.0% 1.0% 0.5% 1.7% FAD2A 5 G150E 18 Null 9.0% 5.1% 17.8% 28.2% 24.9%3.2% 8.9% 1.2% 0.6% 1.1% FAD2A G150E 24 Null 8.7% 5.1% 16.4% 27.4% 26.0%2.2% 10.9% 1.1% 0.6% 1.6% FAD2A 7 S229F 12 Null 1 8.6% 5.6% 15.0% 27.8%26.6% 3.4% 8.2% 1.4% 0.7% 2.8% FAD2A 7 S229F 10 Null 1 9.3% 5.7% 12.8%28.1% 25.4% 3.7% 10.1% 1.8% 0.4% 2.7% FAD2A S229F 13 Null 2 7.8% 5.6%14.1% 26.4% 25.6% 3.6% 12.2% 1.5% 0.7% 2.5% FAD2A S229F 14 Null 7.6%4.8% 13.8% 25.0% 28.3% 3.2% 12.6% 1.2% 0.8% 2.6% FAD2B W91* 7 Null 17.6% 3.9% 12.4% 32.4% 23.9% 2.6% 11.5% 1.9% 0.7% 3.1% FAD2B W91* 5 Null1 7.9% 4.2% 12.8% 29.4% 26.1% 2.6% 11.8% 1.7% 0.7% 2.9% FAD2B W91* 10Null 4 8.4% 5.0% 12.1% 28.8% 26.9% 2.8% 11.4% 1.7% 0.8% 2.0% FAD2B W91*13 Null 7.6% 4.8% 13.0% 33.7% 21.9% 2.8% 10.8% 1.8% 0.6% 2.9% FAD2B W91*23 Null 8.3% 5.8% 13.4% 31.9% 21.4% 4.0% 10.6% 1.7% 0.4% 2.6% FAD2B W91*24 Null 9.1% 5.1% 13.1% 32.8% 24.8% 2.2% 8.4% 1.8% 0.3% 2.5% Note:*stands for nonsense mutation; HOMO means the plants are all homozygousmutants at the specified locus. HET means the plants are heterozygousmutants at the specified locus. NULL means there is no mutation at thespecified locus. % means % of FAME composition

TABLE 18a Fatty Acids Composition in selected FAD2 mutants, sorted bygene, Test 4 Seed Geno- gene- Sample type ration gene mutation C16:0C18:0 C18:1 C18:2 C20:0 C18:3 C20:1 C20:2 C20:3 C22:1 2362-Q10 HOM M5FAD2 A G150E 8.0% 4.3% 21.6% 16.2% 2.3% 30.3% 13.4% 0.8% 0.7% 2.2%2362-Q11 HOM M5 FAD2 A G150E 8.0% 3.6% 20.4% 17.1% 1.8% 32.3% 13.1% 0.8%0.7% 2.2% 2362-Q12 HOM M5 FAD2 A G150E 8.3% 3.6% 19.4% 17.9% 1.5% 32.5%13.0% 0.9% 0.7% 2.2% 2362-Q13 HOM M5 FAD2 A G150E 8.2% 4.2% 20.4% 17.3%2.3% 31.0% 13.1% 0.9% 0.6% 2.1% 2826-P1 Het M5 FAD2 A Q44* 7.1% 3.4%16.3% 16.3% 2.2% 35.1% 14.6% 1.2% 1.1% 2.8% 2826-P2 Het M5 FAD2 A Q44*7.7% 4.0% 17.2% 16.4% 2.5% 33.7% 14.1% 1.0% 0.9% 2.5% 2826-P3 Het M5FAD2 A Q44* 8.4% 4.0% 17.8% 19.8% 2.7% 28.9% 12.4% 1.4% 1.2% 3.3%2826-P4 Het M5 FAD2 A Q44* 7.7% 3.9% 15.5% 16.6% 2.5% 34.4% 14.5% 1.2%1.0% 2.6% 3006-R1 HOM M5 FAD2 B W91* 7.7% 3.5% 11.9% 21.5% 2.3% 35.6%12.3% 1.6% 1.1% 2.6% 3006-R2 HOM M5 FAD2 B W91* 7.8% 3.6% 12.1% 22.6%2.3% 33.4% 12.7% 1.6% 1.0% 2.7% 3006-R3 HOM M5 FAD2 B W91* 8.1% 3.5%12.3% 22.5% 2.1% 34.5% 12.0% 1.6% 1.1% 2.4% 3006-R4 HOM M5 FAD2 B W91*7.8% 3.6% 12.7% 22.2% 2.1% 34.4% 12.2% 1.6% 1.0% 2.4% 3489-N2 HOM M4FAD2 B W138* 8.0% 3.4% 11.9% 23.2% 2.5% 31.7% 12.8% 1.9% 1.1% 3.6%3489-N5 HOM M4 FAD2 B W138* 8.2% 3.5% 11.5% 23.7% 2.6% 31.0% 12.8% 2.0%1.0% 3.6% 3489-N9 HOM M4 FAD2 B W138* 7.9% 3.3% 12.3% 22.2% 2.6% 31.0%13.7% 1.9% 1.2% 3.8% 3489-N12 HOM M4 FAD2 B W138* 7.8% 3.4% 11.9% 22.9%2.6% 30.7% 14.0% 2.0% 1.1% 3.8% 3489-N16 HOM M4 FAD2 B W138* 7.8% 3.5%11.9% 22.7% 2.6% 31.2% 13.6% 2.0% 1.1% 3.8% 3702-O2 HOM M4 FAD2 B G150E7.7% 4.2% 12.8% 23.5% 3.0% 31.1% 12.0% 1.8% 1.0% 2.9% 3702-O3 HOM M4FAD2 B G150E 9.7% 5.6% 17.1% 34.2% 3.7% 18.0% 8.2% 1.0% 0.3% 2.0%3702-O4 Het M4 FAD2 B G150E 7.5% 4.4% 11.6% 24.7% 4.5% 31.1% 10.4% 1.8%0.9% 3.2% 3702-O6 HOM M4 FAD2 B G150E 7.8% 4.4% 12.0% 24.4% 3.0% 31.4%11.5% 1.8% 0.9% 2.7% 3702-O7 HOM M4 FAD2 B G150E 8.0% 5.8% 13.6% 25.2%4.2% 29.7% 9.1% 1.4% 0.7% 2.2% 3702-O9 Het M4 FAD2 B G150E 7.1% 4.0%12.1% 23.7% 3.2% 31.2% 12.4% 1.9% 1.0% 3.3% 6490-M1 HOM M4 FAD2 B W91*6.3% 3.2% 13.2% 21.8% 2.3% 32.1% 14.4% 1.8% 1.2% 3.7% 6490-M2 HOM M4FAD2 B W91* 6.1% 2.9% 12.0% 20.2% 2.2% 34.2% 14.8% 2.1% 1.3% 4.1%6490-M3 HOM M4 FAD2 B W91* 6.2% 3.0% 12.5% 20.8% 2.3% 33.5% 14.5% 2.0%1.3% 4.0% 6490-M4 HOM M4 FAD2 B W91* 8.3% 3.2% 12.3% 21.3% 2.3% 32.0%14.0% 1.8% 1.2% 3.6% 6490-M5 HOM M4 FAD2 B W91* 8.0% 2.5% 12.0% 20.5%1.8% 33.8% 14.7% 1.9% 1.3% 3.6% 6490-M10 null M4 FAD2 B W91* 9.0% 2.6%10.9% 18.5% 2.1% 35.5% 14.4% 2.1% 1.4% 3.3% 3284-B11 null M4 FAD2 C W91*8.9% 4.7% 10.1% 22.3% 2.8% 35.8% 10.3% 2.0% 1.2% 2.0% 3284-B12 Het M4FAD2 C W91* 8.6% 5.2% 11.9% 23.2% 3.2% 33.4% 10.2% 1.6% 0.9% 1.7%3284-B13 Het M4 FAD2 C W91* 8.1% 4.4% 11.6% 21.1% 2.7% 36.3% 11.1% 1.7%1.2% 1.9% 3284-B15 null M4 FAD2 C W91* 9.0% 4.2% 9.3% 22.1% 2.9% 36.9%10.5% 1.9% 1.3% 1.9% 3284-B21 Het M4 FAD2 C W91* 8.4% 4.5% 10.6% 20.3%2.9% 36.5% 11.7% 1.7% 1.2% 2.2% 4506-A2 null M4 FAD2 C W87* 7.5% 3.5%11.9% 23.6% 3.0% 30.8% 12.9% 2.1% 1.1% 3.5% 4506-A10 Hom M4 FAD2 C W87*6.9% 3.6% 14.7% 20.7% 2.6% 31.5% 14.1% 1.6% 1.0% 3.3% 4506-A12 Hom M4FAD2 C W87* 7.3% 4.0% 15.9% 20.4% 2.9% 30.0% 14.1% 1.4% 0.9% 3.2%4506-A15 Hom M4 FAD2 C W87* 8.2% 3.3% 7.1% 24.1% 2.5% 33.8% 14.0% 1.9%1.1% 4.1% 4506-A16 null M4 FAD2 C W87* 8.0% 3.5% 12.8% 23.6% 3.0% 30.9%12.5% 1.7% 1.0% 3.1% 4608-C4 Hom M4 FAD2 C G150E 8.8% 3.5% 9.2% 23.3%2.9% 35.0% 11.2% 1.9% 1.2% 2.9% 4608-C12 Hom M4 FAD2 C G150E 9.3% 4.0%10.0% 25.9% 3.0% 31.6% 10.8% 1.9% 1.0% 2.7% 4608-C13 Het M4 FAD2 C G150E9.0% 3.9% 9.2% 25.4% 2.8% 32.8% 11.0% 2.1% 1.1% 2.8% 4608-C15 null M4FAD2 C G150E 8.9% 3.9% 8.9% 26.0% 2.9% 32.7% 10.7% 2.2% 1.1% 2.7%4608-C17 Het M4 FAD2 C G150E 9.0% 3.8% 8.7% 23.6% 3.1% 34.1% 11.1% 2.2%1.3% 3.1% Cs32-1 7.8% 4.7% 12.4% 27.1% 4.3% 25.5% 12.1% 2.0% 0.8% 3.3%Cs32-2 8.0% 4.5% 12.0% 26.7% 3.9% 27.1% 11.8% 2.0% 0.8% 3.2% Cs32-3 8.0%4.1% 12.1% 26.6% 3.7% 27.7% 11.7% 2.1% 0.9% 3.2% Cs32-4 7.9% 3.9% 12.2%26.2% 3.4% 28.7% 11.7% 2.0% 0.9% 3.0% At FAD2_1 5.4% 3.0% 49.9% 4.2%1.5% 10.3% 24.2% 0.0% 0.0% 1.5% At FAD2_2 5.6% 3.6% 50.5% 4.5% 0.0%10.3% 24.1% 0.1% 0.0% 1.4% At FAE1_1 10.3% 4.7% 28.8% 34.2% 1.0% 20.9%0.1% 0.0% 0.0% 0.0% At FAE1_2 10.2% 5.2% 28.7% 33.9% 1.0% 20.9% 0.1%0.0% 0.0% 0.0% Note: *stands for nonsense mutation; Hom means the plantsare all homozygous mutants at the specified locus. Het means the plantsare heterozygous mutants at the specified locus. Null means there is nomutation at the specified locus. % means % of FAME composition Geneindicates in which gene the mutation is located

TABLE 19a Fatty Acids Composition in selected FAE1 mutants, sorted bygene, Test 4 Seed Geno- gener- Sample type ation gene mutation C16:0C18:0 C18:1 C18:2 C20:0 C18:3 C20:1 C20:2 C20:3 C22:1 3395-D10 Hom M4FAE1 A R209* 9.7% 4.2% 13.2% 20.8% 2.4% 33.6% 11.4% 1.6% 1.1% 2.0%3395-D12 Hom M4 FAE1 A R209* 8.3% 4.7% 15.8% 21.0% 2.4% 32.7% 11.0% 1.3%1.0% 1.6% 3395-D13 Hom M4 FAE1 A R209* 7.8% 4.2% 14.1% 20.7% 2.2% 35.7%11.0% 1.5% 1.1% 1.7% 3395-D17 null M4 FAE1 A R209* 9.8% 3.4% 11.7% 20.8%2.1% 34.6% 11.7% 1.8% 1.3% 2.8% 3395-D18 null M4 FAE1 A R209* 7.4% 4.3%14.0% 21.4% 2.5% 33.5% 11.6% 1.6% 1.2% 2.6% 3395-D19 null M4 FAE1 AR209* 7.7% 3.5% 14.2% 20.2% 2.4% 33.5% 12.7% 1.6% 1.1% 3.1% 3395-D20 HomM4 FAE1 A R209* 7.8% 4.4% 14.8% 21.4% 2.4% 33.8% 11.3% 1.4% 1.0% 1.7%6386-F1 Het M4 FAE1 A G221D 11.1% 5.0% 10.6% 32.7% 3.6% 23.4% 9.2% 1.7%0.5% 2.1% 6386-F2 HOM M4 FAE1 A G221D 9.2% 4.7% 13.3% 26.8% 2.2% 31.3%9.1% 1.3% 0.7% 1.2% 6386-F7 Hom M4 FAE1 A G221D 8.8% 4.6% 12.7% 26.3%2.4% 32.7% 9.0% 1.5% 0.8% 1.1% 6386-F9 Hom M4 FAE1 A G221D 9.0% 4.4%11.5% 26.8% 2.6% 30.9% 10.2% 1.8% 0.9% 1.9% 6386-F13 null M4 FAE1 AG221D 8.9% 4.6% 12.7% 25.4% 2.3% 33.6% 9.0% 1.4% 0.8% 1.2% 6386-F15 nullM4 FAE1 A G221D 8.1% 4.2% 11.2% 25.3% 3.4% 30.1% 11.8% 1.9% 1.0% 2.9%6386-F19 Het M4 FAE1 A G221D 8.2% 4.2% 11.4% 24.8% 2.7% 32.8% 10.9% 1.8%1.0% 2.2% 4687-I4 HOM M4 FAE1 B H301Y 7.1% 2.6% 14.6% 20.7% 1.2% 41.0%9.2% 1.3% 1.1% 1.3% 4687-I10 null M4 FAE1 B H301Y 7.3% 3.2% 14.5% 21.0%1.8% 37.8% 10.5% 1.4% 1.1% 1.5% 4687-I11 HOM M4 FAE1 B H301Y 7.7% 3.2%15.9% 21.8% 1.4% 38.9% 8.2% 1.1% 0.9% 0.9% 4687-I14 null M4 FAE1 B H301Y7.6% 3.6% 16.0% 21.6% 1.8% 34.1% 11.5% 1.4% 1.0% 1.5% 4687-I17 HOM M4FAE1 B H301Y 7.2% 3.0% 14.7% 19.6% 1.3% 43.0% 8.2% 1.1% 1.0% 0.9%5343-H6 null M4 FAE1 B S281F 7.6% 3.5% 11.5% 19.6% 2.5% 36.5% 12.6% 1.8%1.4% 3.0% 5343-H7 null M4 FAE1 B S281F 7.8% 3.6% 11.6% 20.1% 2.6% 35.7%12.5% 1.8% 1.2% 3.1% 5343-H10 HOM M4 FAE1 B S281F 7.9% 4.0% 13.2% 22.9%2.3% 34.5% 10.6% 1.5% 1.0% 2.1% 5343-H14 HOM M4 FAE1 B S281F 8.3% 3.2%11.2% 20.6% 1.8% 39.3% 10.6% 1.7% 1.3% 2.1% 5343-H15 HOM M4 FAE1 B S281F8.1% 4.0% 12.0% 22.1% 2.2% 36.5% 10.4% 1.6% 1.1% 2.0% 5343-H16 HOM M4FAE1 B S281F 7.9% 3.0% 11.0% 19.4% 1.7% 40.6% 10.9% 1.8% 1.5% 2.3%5951-G1 HOM M4 FAE1 B Q142* 8.7% 3.6% 14.1% 22.4% 1.1% 43.0% 4.9% 0.8%0.7% 0.7% 5951-G2 HOM M4 FAE1 B Q142* 8.1% 3.8% 14.4% 22.3% 1.4% 40.7%6.6% 0.9% 0.8% 0.9% 5951-G3 HOM M4 FAE1 B Q142* 7.9% 3.1% 11.3% 20.0%2.1% 39.0% 11.0% 1.7% 1.5% 2.4% 5951-G4 HOM M4 FAE1 B Q142* 9.4% 3.3%14.3% 23.8% 0.9% 44.0% 3.0% 0.5% 0.5% 0.3% 5951-G5 HOM M4 FAE1 B Q142*8.0% 2.8% 10.5% 20.4% 2.2% 39.0% 11.4% 1.7% 1.4% 2.7% 6476-K2 Hom M4FAE1 C R209* 7.8% 3.2% 9.2% 23.2% 2.2% 38.7% 9.4% 1.9% 1.3% 2.9% 6476-K4HOM M4 FAE1 C R209* 7.3% 3.8% 10.6% 23.1% 2.4% 38.3% 9.1% 1.7% 1.2% 2.3%6476-K6 HOM M4 FAE1 C R209* 8.3% 4.0% 10.2% 22.9% 2.5% 36.3% 9.8% 1.9%1.2% 2.9% 6476-K7 null M4 FAE1 C R209* 7.1% 3.6% 9.8% 23.6% 3.0% 32.1%13.0% 2.5% 1.2% 4.0% 6476-K15 HOM M4 FAE1 C R209* 7.5% 4.0% 11.2% 21.6%2.2% 35.6% 11.7% 1.9% 1.2% 3.0% Cs32-1 7.8% 4.7% 12.4% 27.1% 4.3% 25.5%12.1% 2.0% 0.8% 3.3% Cs32-2 8.0% 4.5% 12.0% 26.7% 3.9% 27.1% 11.8% 2.0%0.8% 3.2% Cs32-3 8.0% 4.1% 12.1% 26.6% 3.7% 27.7% 11.7% 2.1% 0.9% 3.2%Cs32-4 7.9% 3.9% 12.2% 26.2% 3.4% 28.7% 11.7% 2.0% 0.9% 3.0% At FAE1_110.3% 4.7% 28.8% 34.2% 1.0% 20.9% 0.1% 0.0% 0.0% 0.0% At FAE1_2 10.2%5.2% 28.7% 33.9% 1.0% 20.9% 0.1% 0.0% 0.0% 0.0% Note: *stands fornonsense mutation; Hom means the plants are all homozygous mutants atthe specified locus. Het means the plants are heterozygous mutants atthe specified locus. Null means there is no mutation at the specifiedlocus. % means % of FAME composition Gene indicates in which gene themutation is located

Example 13 Fatty Acids Composition in Plants with Multiple Mutations inFAD2 and/or FAE1 Genes

To further increase the oleic acid (18:1) level and/or yield and improveCamelina seed oil quality, mutations in one or more copies of FAD2 genesand/or one or more copies of FAE1 genes are integrated together tocreate mutant plants with double, triple, quadruple et al. mutations.Such mutants can be created by classic breeding methods. Table 20 belowshows a list of non-limiting examples of such mutants.

TABLE 20 Plants with more than one mutation in Fatty Acid SynthesisGenes Genotype Plant ID FAD2 A FAD2 B FAD2 C FAE1 A FAE1 B FAE1 C A1HOMO HOMO NULL NULL NULL NULL A2 HOMO NULL HOMO NULL NULL NULL A3 NULLHOMO HOMO NULL NULL NULL A4 HOMO HOMO HOMO NULL NULL NULL A5 NULL NULLNULL HOMO HOMO NULL A6 NULL NULL NULL HOMO NULL HOMO A7 NULL NULL NULLNULL HOMO HOMO A8 NULL NULL NULL HOMO HOMO HOMO A9 HOMO NULL NULL HOMONULL NULL A10 HOMO NULL NULL HOMO HOMO NULL A11 HOMO NULL NULL HOMO HOMOHOMO A12 HOMO HOMO NULL HOMO NULL NULL A13 HOMO HOMO NULL HOMO HOMO NULLA14 HOMO HOMO NULL HOMO HOMO HOMO A15 HOMO HOMO HOMO HOMO NULL NULL A16HOMO HOMO HOMO HOMO HOMO NULL A17 HOMO HOMO HOMO HOMO HOMO HOME Note:HOMO means the plants are all homozygous mutants at the specified locus.NULL means there is no mutation at the specified locus.

Fatty acid compositions in these mutants are then analyzed by gaschromatography (GC). The results will show that one or more of thesemutants produce seed oil with higher oleic acid (18:1) levels and/orlower VLCFA levels when compared to Cs32 control plants or to one ormore single mutants that have only one mutation in a FAD2 gene and/or aFAE1 gene.

Thus, mutations in more than one FAD2 and/or FAE1 genes further increaseoleic acid (18:1) levels and/or lower VLCFA levels, and improve Camelinaseed oil quality.

Example 14 Fatty Acids Composition in RNAi Transgenic Camelina Plants

As described in the present invention, RNAi technology can be used todisrupt one or more fatty acid synthesis genes (e.g., FAD2, FAE1, andother genes) in Camelina to obtain an increase in oleic acid (18:1)and/or a decrease in VLCFA in the seed oil as measured by relativepercent or absolute yield. The advantage of this method is that an RNAiexpression vector can contain a double strand RNA that simultaneouslysuppresses one or more homologous genes. This is extremely helpful inCamelina as the inventors proved it is an allohexaploid species.

Using RNAi technology to knock down expression of all FAD2 genes and/orall FAE1 genes may be more convenient than classic breeding method.Whereas both sense- and antisense-mediated gene silencing have provenfruitful for PTGS in plant cells, RNAi induction can be more efficientlyachieved by specialized expression cassettes that produceself-complementary hairpin (hp)-like RNA molecules. Such cassettestypically include plant promoter and terminator sequences that controlthe expression of two inversely repeated sequences of the target genesthat are separated by a specific spacer.

Upon delivery to plant cells, expression of an RNAi cassette will resultin a dsRNA molecule composed of two distinct regions: a single-strandedloop, encoded by the spacer region, and a double-stranded stem, encodedby the inverted repeats. It is the stem region that is used as asubstrate by the dicer, but, because the spacer itself can potentiallybe recognized as a substrate as well, intron sequences are often used inthe construction of such RNAi vectors (e.g. Meyer et al., 2004, Vectorsfor RNAi technology in poplar. Plant Biol (Stuttg) 6: 100-103). Thesevectors include, but are not limited to, pHANNIBAL, pHANNIBAL,pHELLSGATE, pSAT, pCAMBIA, pGREEN, et al. RNAi (or hpRNA) plantexpression can potentially be delivered to plant cells by various meansof transformation but are typically used by incorporating into binaryplasmids to be delivered to plant cells by Agrobacterium-mediatedtransformation.

Fatty acid synthesis genes that are potential targets include, any oneof FAD2 genes and/or any one of FAE1 genes as provided in the presentinvention, or alternatively along with any other genes involved inCamelina fatty acid synthesis as described herein or elsewhere.

A non-limiting example of using RNAi technology to suppress CamelinaFAD2 genes is described below. A complete hpRNA expression cassette iscomposed of four distinct regions: a promoter and terminator sequence,the ChsA intron sequence, and a dual MCS. The dual MCS results fromcloning of the ChsA intron sequence into pSAT6-MCS and dividing theoriginal MCS into two new, distinct regions, designated MCS-I andMCS-II, which contain the following unique restriction endonucleaserecognition sites: NcoI, BspEI, BglII, XhoI, SacI, and EcoRI in MCS-Iand PstI, SalI, KpnI, SacII, ApaI, XmaI, SmaI, BamHI, and XbaI inMCS-II. The two MCS regions allow the successive cloning of the targetgene sequence in reverse orientation and assembly of a hpRNA sequence.In pSAT6.35S.RNAi, expression of hpRNA is controlled by tandem CaMV 35Spromoter (35SP) and CaMV 35S terminator (35ST), conferring a completeexpression cassette. In pSAT6.Napin.RNAi, expression of hpRNA iscontrolled by Napin plant seed-specific promoter. hpRNA designedaccording to conserved, specific 19 to 29, 19 to 27, or 19 to 21polynucleotides of FAD2 A, FAD2 B, and FAD2 C genes, which does notshare homology to other genes, are introduced into either pSAT6.35S.RNAior pSAT6.Napin.RNA vector to make the final RNAi construct. Suchconserved, specific 15-21 polynucleotides sequences can be designed byone of ordinary skill in the art based on FAD2 genes disclosed in thepresent invention and known Camelina non-FAD2 gene sequences depositedin the GenBank.

Further, pSAT6.35S.RNAi or pSAT6.Napin.RNA vector containing the hpRNAtargeting FAD2 genes is transformed into Camelina plant using the methoddescribed in WO2009/117555, and positive transformants are selected.Northern blot or qPCR is used to verify if one or more FAD2 genes aresuppressed in the transformants. The transgenic lines with the mostefficient suppression in all FAD2 genes are subjected to fatty acidcomposition analysis by GC, and the results indicate such transgenicCamelina plants have an increased oleic acid (18:1) level and/or reducedpolyunsaturated fatty acids level in the seed oil compared to transgenicCamelina plants with empty control vector.

In addition, another pSAT6.35S.RNAi or pSAT6.Napin.RNA vector containingthe hpRNA targeting FAE1 genes is transformed into Camelina plant usingthe method described in WO2009/117555, and positive transformants areselected. Northern blot or qPCR is used to verify if one or more FAE1genes are suppressed in the transformants. The transgenic lines with themost efficient suppression in all FAE1 genes are subjected to fatty acidcomposition analysis by GC, and the results indicate such transgenicCamelina plants have a decreased long chain fatty acid level, and/orreduced long chain polyunsaturated fatty acids level in the seed oilcompared to transgenic Camelina plants with empty control vector.

Example 15 Fatty Acid Composition in Camelina Plants Having SuppressedFAD2 and/or FAE1 Gene Functions in Combination with Overexpression orSuppression of Other Non-FAD and Non-FAE Fatty Acid Synthesis Genes

Other fatty acid synthesis enzymes may be manipulated in the fatty acidsynthesis pathways to increase the amount of oleic acid (18:1) ordecrease the amount of palmitic acid (16:0) to create Camelina oil withfatty acid profiles optimal for biodiesel production. Lower amounts of16:0 saturated fatty acid and higher amounts of 18:1 monounsaturatedfatty acid is desirable for a good balance of proper cetane number,cloud point, oxidative stability, and reduced NOx emissions, asmentioned in the Background and Example 9.

Three key enzymes regulate the amount of 16:0, 18:0 and 18:1 fatty acids(FIG. 13): acyl-acyl carrier protein thioesterase (also known as FATB),β-ketoacyl-acyl carrier protein (ACP) synthase II (KAS II) and Δ-9desaturase. FATB hydrolyzes the fatty acyl group from acyl carrierprotein (ACP) and thus determines the amount and type of fatty acid thatis exported from the plastid. Suppression of FATB leads to a reductionin 16:0 and 18:0 (stearic acid) released to the cytoplasm. KAS IIconverts palmitoyl-ACP (16:0-ACP) to stearoyl-ACP (18:0 ACP), and thusthe overexpression of KAS II leads to an increase in the amount of 16:0being converted to 18:0. Δ-9 desaturase converts 18:0-ACP to oleoyl-ACP(18:1-ACP), and thus the overexpression of Δ-9 desaturase leads to anincrease in the amount of 18:0 being converted to 18:1. Since theproduct of KAS II activity (18:0-ACP) is the substrate for Δ-9desaturase, the overexpression of both KAS II and Δ-9 desaturase willlead to a further decrease in 16:0 and 18:0 and an increase in 18:1.

Camelina lines having suppressed FAD2 and/or FAE1 gene functions, asdescribed in the present invention, obtained either by TILLING ortransgenic means (e.g., antisense, RNAi), may be combined withoverexpression or suppression of the non-FAD and non-FAE genes describedin this example to create new Camelina lines with even greaterpercentages of 18:1 fatty acid and/or lesser percentages of 16:0 and/or18:0 fatty acids compared to lines with only FAD2/FAE1 modifications oronly non-FAD/non-FAE modifications.

For example, Camelina FAD2 and/or FAE1 mutant plants, permutations ofwhich are described in Example 13, may be combined by breeding with aCamelina plant overexpressing KAS II in a seed-specific manner to createa new Camelina line where the amount of 18:1 is higher and the amount of16:0 is lower compared to either parent plant alone. The seed-specificoverexpression of KAS II may also indirectly decrease the amount of 18:2and/or 18:3 polyunsaturated fatty acids.

Alternatively, Camelina FAD2 and/or FAE1 mutant plants may be combinedby breeding with a Camelina plant overexpressing Δ-9 desaturase in aseed-specific manner to create a new Camelina line where the amount of18:1 is higher and the amount of 16:0 is lower compared to either parentplant alone. This combination may also decrease the amount of very longchain fatty acids.

In addition, Camelina FAD2 and/or FAE1 mutant plants may be combined bybreeding with a Camelina plant overexpressing both KAS II and Δ-9desaturase in a seed-specific manner to create a new Camelina line wherethe amount of 18:1 is higher and the amount of 16:0 is lower compared toany of the original parent modifications (FAD2/FAE1 suppression, KAS IIoverexpression or Δ-9 desaturase overexpression) alone.

Optionally, Camelina FAD2 and/or FAE1 mutant plants may be combined bybreeding with a Camelina plant knocked out for FATB function (either byTILLING or transgenic means with a seed-specific promoter) to create anew Camelina line where the amount of 18:1 is higher and the amount of16:0 is lower compared to either parent plant alone. Arabidopsis FATBknockout plants are compromised in growth and produce less viable seeds(Bonaventure et al, The Plant Cell, Vol. 15, 1020-1033, April 2003).This detrimental phenotype may be alleviated in a polyploid likeCamelina, where the presence of multiple copies for a given gene mayallow greater flexibility in manipulating the levels of camelina FATB.Alternatively, the detrimental FATB knockout phenotype may be alleviatedby only suppressing or knocking out FATB function using a FATB antisenseor RNAi construct driven by a seed-specific promoter.

Other combinations of FAD2/FAE1 suppression, KAS II seed-specificoverexpression, Δ-9 desaturase seed-specific overexpression and/or FATBsuppression may be envisioned to obtain Camelina plants with increased18:1 and decreased 16:0 and/or 18:0.

Example 16 Camelina Plants Having Mutations in FAD2 and/or FAE1 Genes inCombination with Overexpression of REV/KRP Genes for Altered Fatty AcidComposition and Increased Seed Yield

The purpose of suppressing Camelina FAD2 and/or FAE1 functions is toobtain an altered fatty acid profile of Camelina oil more suitable forconversion to biodiesel. Another attribute that would contribute toimprovement of the oilseed crop for biofuel purposes would be anincrease in seed yield, either by an increase in total seed number orseed size, in order to increase the amount of oil recovered per unit ofland. Two yield technologies, REV and KRP dominant negative, have beendescribed (US 2008/263727 and US 2007/056058, incorporated by referencein their entireties) that give increased seed yield when overexpressedunder early embryo-specific promoters.

Camelina FAD2 and/or FAE1 mutant plants, permutations of which aredescribed in Example 13, may be combined by breeding with a Camelinaplant overexpressing REV in an early embryo-specific manner to create anew Camelina line with greater seed yield and high 18:1 and/or lowVLCFAs compared to either parent plant alone.

Similarly, Camelina FAD2 and/or FAE1 mutant plants may be combined bybreeding with a Camelina plant overexpressing KRP dominant negative inan early embryo-specific or constitutive manner to create a new Camelinaline with greater seed yield and high 18:1 and/or low VLCFAs compared toeither parent plant alone.

Additionally, Camelina FAD2 and/or FAE1 mutant plants may be combined bybreeding with a Camelina plant overexpressing both REV and KRP dominantnegative in an early embryo-specific (or constitutive for KRP) manner tocreate a new Camelina line with greater seed yield and high 18:1 and/orlow VLCFAs compared to any of the original parent modifications(FAD2/FAE1 suppression, early embryo-specific REV overexpression orembryo-specific or constitutive KRP dominant negative overexpression)alone.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only examples and should not be taken aslimiting the scope of the invention.

Unless defined otherwise, all technical and scientific terms herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Definitions of common terms inmolecular biology may be found in Benjamin Lewin, Genes IX, published byOxford University Press, 2007 (ISBN-10 0131439812); Kendrew et al.(eds.), The Encyclopedia of Molecular Biology, published by BlackwellScience Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.),Molecular Biology and Biotechnology: A Comprehensive Desk Reference,published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); OxfordDictionary of Biochemistry and Molecular Biology, Revised Edition, 2000.Although any methods and materials, similar or equivalent to thosedescribed herein, can be used in the practice or testing of the presentinvention, the preferred methods and materials are described herein.

All publications, patents, and patent publications cited areincorporated by reference herein in their entirety for all purposes.Also incorporated by reference herein are nucleic acid sequences andpolypeptide sequences deposited into the GenBank, which are cited inthis specification.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

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We claim:
 1. An isolated polynucleotide encoding a plant fatty acidsynthesis polypeptide, wherein the isolated polynucleotide encodes aplant fatty acid desaturase (FAD) polypeptide, comprising a sequencesharing at least 93% identity to SEQ ID NO: 1, 2, 3, 45, 46, 48, 51, 54,55, 56, 60, or 61; or the isolated polynucleotide encodes a plant fattyacid elongase (FAE) polypeptide comprising a sequence sharing at least91% identity to SEQ ID NO: 4, 5, 6, 47, 49, 50, 52, 53, 57, 58, 59, 62,or
 63. 2. The isolated FAD polynucleotide of claim 1, wherein thepolynucleotide comprises a sequence selected from the group consistingof SEQ ID NOs 1, 2, 3, 45, 46, 48, 51, 54, 55, 56, 60, and 61; or theisolated FAE polynucleotide of claim 1, wherein the polynucleotidecomprises a sequence selected from the group consisting of SEQ ID NOs 4,5, 6, 47, 49, 50, 52, 53, 57, 58, 59, 62, and
 63. 3. The isolatedpolynucleotide of claim 1, wherein the FAD polynucleotide comprises oneor more mutations listed in Tables 7 to 9; or wherein the FAEpolynucleotide comprises one or more mutations listed in Tables 10 to12.
 4. An isolated FAD polypeptide comprising an amino acid sequencesharing at least 93% identity to a FAD polypeptide encoded by the FADpolynucleotide of claim 1; or an isolated FAE polypeptide comprising anamino acid sequence sharing at least 88% identity to a FAE polypeptideencoded by the FAE polynucleotide of claim
 1. 5. A plant cell, plantpart, plant tissue culture or whole plant comprising at least one FAD2gene comprising at least one mutation that disrupts and/or alters thefunction of the at least one FAD2 gene and/or at least one FAE1 genecomprising at least one mutation that disrupts and/or alters thefunction of the at least one FAE1 gene.
 6. The plant cell, plant part,plant tissue culture or whole plant of claim 5 wherein the FAD2 gene isFAD2 A, FAD2 B and/or FAD2 C; and wherein the FAE1 gene is FAE1 A. FAE1Band/or FAE1 C.
 7. The plant cell, plant part, plant tissue culture orwhole plant of claim 5, wherein the plant is a Camelina plant.
 8. TheCamelina plant cell, plant part, plant tissue culture or whole plant ofclaim 5 further comprising one or more additional genetic changesselected from the group consisting of: (a) one or more non-FAD2,non-FAE1 fatty acid synthesis genes with at least one mutation thatdisrupts and/or alters the function of that gene; (b) a chimeric genecomprising a double stranded RNA region that is both homologous andcomplementary to a region of one or more non-FAD2, non-FAE1 fatty acidsynthesis genes; and (c) a chimeric gene comprising one or morenon-FAD2, non-FAE1 fatty acid synthesis genes, wherein the non-FAD2,non-FAE1 fatty acid synthesis genes are operably linked to anoverexpression promoter.
 9. The plant cell, plant part, tissue cultureor whole plant of claim 8, wherein the additional fatty acid gene isselected from the group consisting of FAD3, a hydroxylase and athioesterase.
 10. The plant cell, plant part, plant tissue culture orwhole plant of claim 5; wherein the mutation is selected from any one ofmutations listed in Tables 7 to 12 for a particular FAD2 or FAE1 gene.11. A method of increasing the activity of a FAD2 and/or FAE1 protein ina Camelina plant cell, plant part, tissue culture or whole plantcomprising transforming the plant cell, plant part, tissue culture orwhole plant with a chimeric gene comprising one FAD2 and/or FAE1 geneencoding the polypeptide of claim 4, or functional variants thereof.